WO2020069656A1

WO2020069656A1 - Method for detecting fish pathogen

Info

Publication number: WO2020069656A1
Application number: PCT/CN2019/108518
Authority: WO
Inventors: 郭村勇; 谢旺儒; 陈台安; 陈子翔; 谢凯如; 萧志奇
Original assignee: 福又达生物科技股份有限公司
Priority date: 2018-10-05
Filing date: 2019-09-27
Publication date: 2020-04-09
Also published as: CN111004865A; TW202014524A; TWI817777B; TW202014525A; CL2021000615A1; TW202132574A; EP3862443A4; TW202024338A; EP3862443A1; WO2020069657A1; TWI730432B; TWI732303B; TWI784467B; TWI727444B; CN111004863A; CN111004864A; WO2020069658A1; TW202309278A

Abstract

A method for detecting a fish pathogen, and an oligonucleotide pair for detecting a fish pathogen.

Description

Fish pathogen detection method

Technical field

The invention relates to a method for detecting fish pathogens, in particular to a method for detecting fish pathogens using oligonucleotide pairs.

Background technique

Aquatic animals are one of the important sources of human energy, protein and essential nutrients. The suspicion that a large number of wild aquatic animals are depleted of marine resources has made the aquaculture industry increasingly valued. According to the statistics of the Food and Agriculture Organization of the United Nations, aquaculture's contribution to global fishing and total aquaculture production has gradually increased from 25.7% in 2000 to 46.8% in 2016. However, intensive farming coupled with poor management may cause aquatic animals to be susceptible to disease and death, and cause losses to fishermen. In addition, the diseases of aquatic animals are mostly caused by bacteria or viruses. The treatment methods for bacterial diseases and viral diseases are far from each other. Once misdiagnosed and improperly handled, it will cause huge losses. This shows that intensive monitoring of diseases in fish farms and shrimp farms is an indispensable part of management. The invention provides a quick and convenient method for detecting fish pathogens for industrial use.

Summary of the invention

In one aspect, the present invention relates to a fish pathogen detection method, comprising providing a sample that may contain one or more nucleotide sequences of a fish pathogen; providing an oligonucleotide primer pair, the oligonucleotide primer pair Containing a first primer and a second primer, the oligonucleotide primer pair defines the 5 'end of two complementary strands of a double-stranded target sequence on one or more nucleotide sequences of the pathogen; provides a polymerization Enzyme; mix the sample, the oligonucleotide primer pair, the polymerase, deoxyadenosine triphosphates (dATPs), deoxycytidine triphosphates (dCTPs) in a container, Deoxyguanosine triphosphates (dGTPs) and deoxythymidine triphosphates (dTTPs) to form a polymerase chain reaction (PCR) mixture; by heating at a fixed temperature At the bottom of the container, the PCR mixture is subjected to thermal convection polymerase chain reaction (cPCR) to form a PCR product ; And detecting the PCR product to recognize the double-stranded target sequence.

In certain embodiments, the polymerase chain reaction (PCR) mixture further comprises an oligonucleotide probe comprising a segment complementary to a segment of the double-stranded target sequence Sequence of an oligonucleotide probe, a fluorescent molecule attached to a first position on the oligonucleotide probe, and a fluorescence attached to a second position on the oligonucleotide probe An inhibitory molecule, when the oligonucleotide probe is not hybridized to the segment of the double-stranded target sequence, the fluorescence inhibitory molecule substantially inhibits the fluorescent molecule, and when the oligonucleotide When the probe is hybridized to the segment of the double-stranded target sequence, the fluorescent molecule is not substantially inhibited.

In another aspect, the invention relates to an oligonucleotide pair for detecting fish pathogens, comprising a first primer and a second primer.

In certain embodiments, the oligonucleotide pair further comprises an oligonucleotide probe having an oligonucleotide probe sequence, an appendage to the oligonucleotide A fluorescent molecule at a first position on the acid probe, and a fluorescent inhibitor molecule at a second position attached to the oligonucleotide probe.

In some embodiments, the pathogen is fish respiratory tract and enterovirus (Piscinereovirus, PRV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 10, and SEQ ID NO: 11 and SEQ ID NO: 12.

In certain embodiments, the pathogen is fish respiratory tract and enterovirus (PRV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 1 and SEQ ID NO: 2 The oligonucleotide probe sequence is between the 71st and 95th nucleotides of the L1 segment gene (GenBank accession No. KY429943) of the pathogen fish respiratory tract and enterovirus (PRV) 13 to 25 base pair oligonucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 3.

In certain embodiments, the pathogen is fish respiratory tract and enterovirus (PRV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 4 and SEQ ID NO: 2 The oligonucleotide probe sequence is between the 52nd and 95th nucleotides of the L1 segment gene (GenBank accession No. KY429943) of the pathogen fish respiratory tract and enterovirus (PRV) 13 to 35 base pair oligonucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 3.

In certain embodiments, the pathogen is fish respiratory tract and enterovirus (PRV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 4 and SEQ ID NO: 5 The oligonucleotide probe sequence is between the 52nd and 100th nucleotides of the L1 segment gene (GenBank accession No. KY429943) of the pathogen fish respiratory tract and enterovirus (PRV) 13 to 35 base pair oligonucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 3.

In certain embodiments, the pathogen is fish respiratory tract and enterovirus (PRV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 6 and SEQ ID NO: 7 The oligonucleotide probe sequence is between the 343th and 385th nucleotides of the Sigma 3 protein gene (GenBank accession No. KX844958) of the pathogen fish respiratory tract and enterovirus (PRV) 13 to 35 base pair oligonucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 8.

In certain embodiments, the pathogen is fish respiratory tract and enterovirus (PRV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 9 and SEQ ID NO: 10 The oligonucleotide probe sequence is between the 350th and 380th nucleotides of the Sigma 3 protein gene (GenBank accession No. KX844958) of the pathogen fish respiratory tract and enterovirus (PRV) 13 to 35 base pair oligonucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 8.

In certain embodiments, the pathogen is fish respiratory tract and enterovirus (PRV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 11 and SEQ ID NO: 12 , The oligonucleotide probe sequence is between the 280th and 315th nucleotides of the L1 segment gene (GenBank accession No. KY429943) of the pathogen fish respiratory tract and enterovirus (PRV) 13 to 35 base pair oligonucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 13.

In some embodiments, the pathogen is an infectious pancreatic necrosis virus (IPNV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 14 and SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 21, and SEQ ID NO: 23 and SEQ ID NO: 24.

In certain embodiments, the pathogen is infectious pancreatic necrosis virus (IPNV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 14 and SEQ ID NO: 15 , The oligonucleotide probe sequence is between 2010 and 2057 of the VP5 and polyprotein genes (GenBank accession No. KY548514) of the pathogen infectious pancreatic necrosis virus (IPNV) 13 to 35 base pair oligonucleotides between nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 16.

In certain embodiments, the pathogen is infectious pancreatic necrosis virus (IPNV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 17 and SEQ ID NO: 18 The oligonucleotide probe sequence is between the 2345th and 2408th nucleus of the pathogen infectious pancreatic necrosis virus (IPNV) VP5 and the polyprotein (GenBank accession No. KY548514) 13 to 35 base pair oligonucleotides between nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 19.

In certain embodiments, the pathogen is infectious pancreatic necrosis virus (IPNV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 20 and SEQ ID NO: 21 The oligonucleotide probe sequence is between the 2533th and 2580th nucleus of the pathogen infectious pancreatic necrosis virus (IPNV) VP5 and the polyprotein gene (GenBank accession No. KY548514) 13 to 35 base pair oligonucleotides between nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 22.

In certain embodiments, the pathogen is infectious pancreatic necrosis virus (IPNV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 23 and SEQ ID NO: 24 The oligonucleotide probe sequence is between the 2775th to the 2818th nucleus of the pathogen infectious pancreatic necrosis virus (IPNV) VP5 and the polyprotein gene (GenBank accession No. KY548514) 13 to 35 base pair oligonucleotides between nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 25.

In certain embodiments, the pathogen is an infectious salmon anemia virus (ISAV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID: NO: 26 and SEQ ID: NO: 27, SEQ ID: NO: 29 and SEQ ID: NO: 30, SEQ ID: NO: 32 and SEQ ID: NO: 33, SEQ ID: NO: 35 and SEQ ID: NO: 36, and SEQ ID NO: 37 and SEQ ID NO: 38.

In certain embodiments, the pathogen is an infectious salmon anemia virus (ISAV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 26 and SEQ ID NO: 27, The oligonucleotide probe sequence is between the 282th to the ORF2 of the Open Reading Frame (ORF) 1 and ORF2 genes (GenBank accession No. KX424589) of the pathogen infectious salmon anemia virus (ISAV) 13 to 35 base pair oligonucleotides between the 328th nucleotide; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 28.

In certain embodiments, the pathogen is an infectious salmon anemia virus (ISAV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 29 and SEQ ID NO: 30, The oligonucleotide probe sequence is a 13 to 13 nucleotide between the 79th and 121st nucleotides of the ORF1 and ORF2 genes (GenBank accession No. KX424589) of the pathogen infectious salmon anemia virus (ISAV) An oligonucleotide of 35 base pairs; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 31.

In certain embodiments, the pathogen is an infectious salmon anemia virus (ISAV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 32 and SEQ ID NO: 33, The oligonucleotide probe sequence is a 13 to nucleotide number 244 to 273 between the ORF1 and ORF2 genes (GenBank accession No. KX424589) of the pathogen infectious salmon anemia virus (ISAV) An oligonucleotide of 30 base pairs; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 34.

In certain embodiments, the pathogen is an infectious salmon anemia virus (ISAV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 35 and SEQ ID NO: 36, The oligonucleotide probe sequence is a 13 to nucleotide number between 237 and 272 nucleotides between the ORF1 and ORF2 genes (GenBank accession No. KX424589) of the pathogen infectious salmon anemia virus (ISAV) An oligonucleotide of 35 base pairs; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 34.

In certain embodiments, the pathogen is an infectious salmon anemia virus (ISAV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 37 and SEQ ID NO: 38, The oligonucleotide probe sequence is a 13 to 286 nucleotide between the ORF1 and ORF2 genes (GenBank accession No. KX424589) of the pathogen infectious salmon anemia virus (ISAV) An oligonucleotide of 35 base pairs; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 34.

In certain embodiments, the pathogen is Piscine myocarditis virus (PMCV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 39 and SEQ ID NO: 40, SEQ ID NO: 42 and SEQ ID NO: 43, SEQ ID NO: 45 and SEQ ID NO: 46, SEQ ID NO: 48 and SEQ ID NO: 49, SEQ ID NO: 51 and SEQ ID NO: 49, and SEQ ID NO: 52 and SEQ ID NO: 49.

In certain embodiments, the pathogen is fish myocarditis virus (PMCV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 39 and SEQ ID NO: 40, the The oligonucleotide probe sequence is an oligo between 13 to 35 base pairs between the 6266th to 6306th nucleotides of the genome of the pathogen fish myocarditis virus (PMCV) (GenBank accession No. HQ339954) Nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 41.

In certain embodiments, the pathogen is fish myocarditis virus (PMCV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 42 and SEQ ID NO: 43, the The oligonucleotide probe sequence is an oligo between 13 to 35 base pairs between the 5632th to 5672nd nucleotides of the genome (GenBank accession No. HQ339954) of the pathogenic fish myocarditis virus (PMCV) Nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 44.

In certain embodiments, the pathogen is fish myocarditis virus (PMCV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 45 and SEQ ID NO: 46, the The oligonucleotide probe sequence is a 13 to 35 base pair oligo between the 5693th to 5747th nucleotides of the genome (GenBank accession No. HQ339954) of the pathogen fish myocarditis virus (PMCV) Nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 47.

In certain embodiments, the pathogen is fish myocarditis virus (PMCV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 48 and SEQ ID NO: 49, the The oligonucleotide probe sequence is an oligo with 13 to 35 base pairs between the 6294 to 6331 nucleotides of the genome of the pathogen fish myocarditis virus (PMCV) (GenBank accession No. HQ339954) Nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 50.

In certain embodiments, the pathogen is fish myocarditis virus (PMCV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 51 and SEQ ID NO: 49, the The oligonucleotide probe sequence is an oligo between 13 to 35 base pairs between the 6271th to 6331st nucleotides of the genome of the pathogen fish myocarditis virus (PMCV) (GenBank accession No. HQ339954) Nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 50.

In certain embodiments, the pathogen is fish myocarditis virus (PMCV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 52 and SEQ ID NO: 49, the The oligonucleotide probe sequence is an oligo between 13 and 35 base pairs between the 6290th and 6331st nucleotides of the genome of the pathogen fish myocarditis virus (PMCV) (GenBank accession No. HQ339954) Nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 50.

In certain embodiments, the pathogen is Salmonid alphavirus (SAV), and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 53 And SEQ ID NO: 54, SEQ ID NO: 56 and SEQ ID NO: 57, SEQ ID NO: 59 and SEQ ID NO: 60, SEQ ID NO: 62 and SEQ ID NO: 63, and SEQ ID NO: 65 and SEQ ID NO: 66.

In certain embodiments, the pathogen is salmonella virus (SAV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 53 and SEQ ID NO: 54, the The oligonucleotide probe sequence is an oligo with 13 to 35 base pairs between the 611th to 653rd nucleotides of the genome of the pathogen Salmon Alpha Virus (SAV) (GenBank accession No. KC122926) Nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 55.

In certain embodiments, the pathogen is salmonella virus (SAV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 56 and SEQ ID NO: 57, the The oligonucleotide probe sequence is an oligo between 13 and 35 base pairs between the 145th and 181st nucleotides of the genome (GenBank accession No. KC122926) of the pathogen Salmon Alpha Virus (SAV) Nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 58.

In certain embodiments, the pathogen is salmonella virus (SAV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 59 and SEQ ID NO: 60, the The oligonucleotide probe sequence is an oligo between 13 and 35 base pairs between the 2691th to the 2761st nucleotides in the genome (GenBank accession No. KC122926) of the pathogen Salmon Alpha Virus (SAV) Nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 61.

In certain embodiments, the pathogen is salmonella virus (SAV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 62 and SEQ ID NO: 63, the The oligonucleotide probe sequence is an oligo with 13 to 35 base pairs between the 1032th to 1075th nucleotides of the genome (GenBank accession No. KC122926) of the pathogen Salmon Alpha Virus (SAV) Nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 64.

In certain embodiments, the pathogen is salmonella virus (SAV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 65 and SEQ ID NO: 66, the The oligonucleotide probe sequence is an oligo with 13 to 35 base pairs between the 969th to 1011th nucleotides of the genome (GenBank accession No. KC122926) of the pathogen Salmon Alpha Virus (SAV) Nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 67.

In certain embodiments, the pathogen is Neoparamoeba perurans, and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO : 68 and SEQ ID NO: 69, SEQ ID NO: 71 and SEQ ID NO: 72, SEQ ID NO: 74 and SEQ ID NO: 75, and SEQ ID NO: 77 and SEQ ID NO: 78.

In certain embodiments, the pathogen is N. perurans, and when the sequence combination of the first primer and the second primer is SEQ ID NO: 68 and SEQ ID NO : At 69, the oligonucleotide probe sequence is an 18S ribosomal ribonucleic acid (ribosomal RNA, rRNA) gene (GenBank accession No. KU985058) between the pathogen N. perurans ) From 462 to 520th nucleotides between 13 and 35 base pairs of oligonucleotides; in certain preferred embodiments, the oligonucleotide probe sequence such as SEQ ID NO: 70 shown.

In certain embodiments, the pathogen is N. perurans, and when the sequence combination of the first primer and the second primer is SEQ ID NO: 71 and SEQ ID NO : At 72, the oligonucleotide probe sequence is between the 355th and 405th nuclei of the 18S rRNA gene (GenBank accession No. KU985058) of the pathogen N. perurans 13 to 35 base pair oligonucleotides between nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 73.

In certain embodiments, the pathogen is N. perurans, and when the sequence combination of the first primer and the second primer is SEQ ID NO: 74 and SEQ ID NO : At 75, the oligonucleotide probe sequence is between the 234th and 281st nuclei of the 18S rRNA gene (GenBank accession No. KU985058) of the pathogen N. perurans Oligonucleotides of 13 to 35 base pairs between nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 76.

In certain embodiments, the pathogen is N. perurans, and when the sequence combination of the first primer and the second primer is SEQ ID NO: 77 and SEQ ID NO : 78, the oligonucleotide probe sequence is between the 357th and 380th nuclei of the 18S rRNA gene (GenBank accession No. KU985058) of the pathogen N. perurans 13 to 24 base pair oligonucleotides between nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 79.

In certain embodiments, the pathogen is Candidatus Branchiomonas cysticola (CBc) and the sequence combination of the first primer and the second primer is selected from the group consisting of SEQ ID NO: 80 and SEQ ID NO : 81, and SEQ ID NO: 83 and SEQ ID NO: 84.

In certain embodiments, the pathogen is Ca.B. cysticola (CBc), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 80 and SEQ ID NO: 81, The oligonucleotide probe sequence is a nucleotide number 1068 to 1125 between the 16S ribosomal RNA (rRNA) gene (GenBank accession No. JQ723599) of the pathogen Ca.B. cysticola Oligonucleotides between 13 and 35 base pairs; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 82.

In certain embodiments, the pathogen is Ca.B. cysticola (CBc), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 83 and SEQ ID NO: 84, The oligonucleotide probe sequence is a 13 to 30 bases between the 1212th to 1241st nucleotides of the 16S rRNA gene (GenBank accession No. JQ723599) of the pathogen Ca.B. cysticola Pair of oligonucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 85.

In certain embodiments, the pathogen is Desmozoon lepeophtherii (Des) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 86 and SEQ ID NO: 87, SEQ ID NO: 89 and SEQ ID NO: 90, SEQ ID NO: 92 and SEQ ID NO: 93, SEQ ID NO: 95 and SEQ ID NO: 97, and SEQ ID NO: 96 and SEQ ID NO: 97.

In certain embodiments, the pathogen is Desmozoon lepeophtherii (Des), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 86 and SEQ ID NO: 87, The oligonucleotide probe sequence is a 967th to 1010th nucleoside between the 16S ribosomal RNA (rRNA) gene (GenBank accession No. JQ723599) of the pathogen Microsporidium D. lepeophtherii 13 to 35 base pair oligonucleotides between acids; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 88.

In some embodiments, the pathogen is Desmozoon lepeophtherii (Des), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 89 and SEQ ID NO: 90, The oligonucleotide probe sequence is a 13 to 35 base between the 1246th to 1285th nucleotides of the 16S rRNA gene (GenBank accession No. JQ723599) of the pathogen Microsporidium D. lepeophtherii Oligonucleotides in base pairs; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 91.

In certain embodiments, the pathogen is Desmozoon lepeophtherii (Des), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 92 and SEQ ID NO: 93, The oligonucleotide probe sequence is a 13 to 35 base between 1366 to 1414 nucleotides of the 16S rRNA gene (GenBank accession No. JQ723599) of the pathogen Microsporidium D. lepeophtherii Oligonucleotides in base pairs; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 94.

In certain embodiments, the pathogen is Desmozoon lepeophtherii (Des), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 95 and SEQ ID NO: 97, The oligonucleotide probe sequence is a 13 to 35 base between the 815th to 859th nucleotides of the 16S rRNA gene (GenBank accession No. JQ723599) of the pathogen Microsporidium D. lepeophtherii Oligonucleotides in base pairs; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 98.

In certain embodiments, the pathogen is Desmozoon lepeophtherii (Des), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 96 and SEQ ID NO: 97, The oligonucleotide probe sequence is a 13 to 35 base between the 817th to 859th nucleotides of the 16S rRNA gene (GenBank accession No. JQ723599) of the pathogen Microsporidium D. lepeophtherii Oligonucleotides in base pairs; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 98.

In certain embodiments, the pathogen is Salmon poxvirus (SGPV) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 99 and SEQ ID NO: 100, SEQ ID NO: 102 and SEQ ID NO: 103, SEQ ID NO: 105 and SEQ ID NO: 106, and SEQ ID NO: 108 and SEQ ID NO: 109.

In certain embodiments, the pathogen is salmon parrot virus (SGPV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 99 and SEQ ID NO: 100, the The oligonucleotide probe sequence is a 13 to 35 base pair between the 99272th to 99318th nucleotides of the genome of the pathogen salmon parrot virus (SGPV) (GenBank accession No. KT159937) Oligonucleotide; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 101.

In certain embodiments, the pathogen is salmon parrot virus (SGPV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 102 and SEQ ID NO: 103, the The oligonucleotide probe sequence is a 13 to 27 base pair between the 123213th to 123239th nucleotides of the genome (GenBank accession No. KT159937) of the pathogen salmon parrot virus (SGPV) Oligonucleotide; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 104.

In certain embodiments, the pathogen is salmon parrot virus (SGPV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 105 and SEQ ID NO: 106, the The oligonucleotide probe sequence is a 13 to 30 base pair between the 123202th to 123232th nucleotides of the genome of the pathogen salmon parrot virus (SGPV) (GenBank accession No. KT159937) Oligonucleotide; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 107.

In certain embodiments, the pathogen is salmon parrot virus (SGPV), and when the sequence combination of the first primer and the second primer is SEQ ID NO: 108 and SEQ ID NO: 109, the The oligonucleotide probe sequence is a 13 to 35 base pair between the 123556th to 123610th nucleotides of the genome (GenBank accession No. KT159937) of the pathogen salmon parrot virus (SGPV) Oligonucleotide; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 110.

In certain embodiments, the pathogen is Piscirickettsia (salmonis) and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 111 and SEQ ID NO: 113, SEQ ID NO: 114 and SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117, SEQ ID NO: 119 and SEQ ID NO: 120, and SEQ ID NO: 122 and SEQ ID NO: 123.

In certain embodiments, the pathogen is P. salmonis, and when the sequence combination of the first primer and the second primer is SEQ ID NO: 111 and SEQ ID NO: 112 At this time, the oligonucleotide probe sequence is a 952th part of the 16S ribosomal ribonucleic acid (ribosomal RNA, rRNA) gene (GenBank accession No. AY498634) of the pathogen P. salmonis 13 to 35 base pair oligonucleotides up to the 1065th nucleotide; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 113.

In certain embodiments, the pathogen is P. salmonis, and when the sequence combination of the first primer and the second primer is SEQ ID NO: 114 and SEQ ID NO: 115 At this time, the oligonucleotide probe sequence is between the 1014th and 1050th nucleotides of the 16S rRNA gene (GenBank accession No. AY498634) of the pathogen P. salmonis 13 to 35 base pairs of oligonucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 113.

In certain embodiments, the pathogen is P. salmonis, and when the sequence combination of the first primer and the second primer is SEQ ID NO: 116 and SEQ ID NO: 117 At this time, the oligonucleotide probe sequence is between the 315th and 331rd nucleotides of the 16S rRNA gene (GenBank accession No. AY498634) of the pathogen P. salmonis 13 to 17 base pairs of oligonucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 118.

In some embodiments, the pathogen is P. salmonis, and when the sequence combination of the first primer and the second primer is SEQ ID NO: 119 and SEQ ID NO: 120 At this time, the oligonucleotide probe sequence is between the 529th and 545th nucleotides of the 16S rRNA gene (GenBank accession No. AY498634) of the pathogen P. salmonis 13 to 17 base pairs of oligonucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 121.

In some embodiments, the pathogen is P. salmonis, and when the sequence combination of the first primer and the second primer is SEQ ID NO: 122 and SEQ ID NO: 123 When the oligonucleotide probe sequence is between 1181 and 1200 nucleotides of the 16S rRNA gene (GenBank accession No. AY498634) of the pathogen P. salmonis (P. salmonis) 13 to 20 base pairs of oligonucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 124.

In certain embodiments, the pathogen is Renibacterium salmoninarum and the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 126 and SEQ ID NO: 128, SEQ ID NO: 127 and SEQ ID NO: 128, SEQ ID NO: 130 and SEQ ID NO: 131, SEQ ID NO: 133 and SEQ ID NO: 134, and SEQ ID NO: 136 and SEQ ID NO : 137.

In some embodiments, the pathogen is R. salmoninarum, and when the sequence combination of the first primer and the second primer is SEQ ID NO: 125 and SEQ ID NO: 128, The oligonucleotide probe sequence is from the 1035th to the 35th of the 57KD extracellular protein precursor (ECP) gene (GenBank accession No. Z12174) of the pathogen R. salmoninarum 13 to 31 base pair oligonucleotides between 1065 nucleotides; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 129.

In certain embodiments, the pathogen is R. salmoninarum, and when the sequence combination of the first primer and the second primer is SEQ ID NO: 126 and SEQ ID NO: 128, The oligonucleotide probe sequence is a 1024 to 1065 nucleoside between the 57KD extracellular protein precursor (ECP) gene (GenBank accession No. Z12174) of the pathogen R. salmoninarum 13 to 35 base pair oligonucleotides between acids; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 129.

In some embodiments, the pathogen is R. salmoninarum, and when the sequence combination of the first primer and the second primer is SEQ ID NO: 127 and SEQ ID NO: 128, The oligonucleotide probe sequence is a 1037th to 1065th nucleoside between the 57KD extracellular protein precursor (ECP) gene (GenBank accession No. Z12174) of the pathogen R. salmoninarum 13 to 29 base pair oligonucleotides between acids; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 129.

In certain embodiments, the pathogen is R. salmoninarum, and when the sequence combination of the first primer and the second primer is SEQ ID NO: 130 and SEQ ID NO: 131, The oligonucleotide probe sequence is a nucleoside 782 to 806 between the 57KD extracellular protein precursor (ECP) gene (GenBank accession No. Z12174) of the pathogen R. salmoninarum 13 to 25 base pair oligonucleotides between acids; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 132.

In some embodiments, the pathogen is R. salmoninarum, and when the sequence combination of the first primer and the second primer is SEQ ID NO: 133 and SEQ ID NO: 134, The oligonucleotide probe sequence is between the 880th and 964th nucleosides of the 57KD extracellular protein precursor (ECP) gene (GenBank accession No. Z12174) of the pathogen R. salmoninarum 13 to 35 base pair oligonucleotides between acids; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 135.

In certain embodiments, the pathogen is R. salmoninarum, and when the sequence combination of the first primer and the second primer is SEQ ID NO: 136 and SEQ ID NO: 137, The oligonucleotide probe sequence is between the 646th and 738th nucleosides of the 57KD extracellular protein precursor (ECP) gene (GenBank accession No. Z12174) of the pathogen R. salmoninarum 13 to 35 base pair oligonucleotides between acids; in certain preferred embodiments, the oligonucleotide probe sequence is shown in SEQ ID NO: 138.

All technical and scientific terms in this specification, unless otherwise defined, are in the field and have the meaning commonly understood by those skilled in the art.

The present invention is exemplified and illustrated by the following examples, but the present invention is not limited by the following examples.

detailed description

In one aspect, the present invention relates to a fish pathogen detection method. In some embodiments, the method is polymerase chain reaction (PCR). In certain embodiments, the method is reverse-transcription polymerase chain reaction (RT-PCR). In certain embodiments, the method is thermal convection polymerase chain reaction (cPCR). In certain embodiments, the method is real-time polymerase chain reaction (real-time polymerase chain reaction, real-time PCR).

In one aspect, the present invention relates to a fish pathogen detection method, including:

Provide a sample that may contain one or more nucleotide sequences of fish pathogens;

An oligonucleotide primer pair is provided. The oligonucleotide primer pair includes a first primer and a second primer. The oligonucleotide primer pair is defined in one or more nucleotide sequences of the pathogen 5 'end of the two complementary strands of the previous double strand target sequence;

Provide a polymerase;

Mix the sample, the oligonucleotide primer pair, the polymerase, deoxyadenosine triphosphate (dATPs), deoxycytidine triphosphate (dCTPs), deoxyguanosine triphosphate (dGTPs) in a container , And deoxythymidine triphosphates (dTTPs) to form a polymerase chain reaction (PCR) mixture;

By heating the bottom of the container at a fixed temperature, subjecting the PCR mixture to thermal convection polymerase chain reaction (cPCR) to form a PCR product; and

The PCR product is detected to identify the double-stranded target sequence.

In another aspect, the invention relates to a fish pathogen detection method, comprising:

An oligonucleotide probe is provided, the oligonucleotide probe comprising an oligonucleotide probe sequence complementary to a segment of the double-stranded target sequence, an probe attached to the oligonucleotide probe A fluorescent molecule at a first position on the needle, and a fluorescent inhibitor molecule at a second position attached to the oligonucleotide probe, when the oligonucleotide probe is not hybridized to the double When the segment of the target sequence is stranded, the fluorescence suppression molecule substantially suppresses the fluorescent molecule, and when the oligonucleotide probe is hybridized to the segment of the double-stranded target sequence, the The fluorescent molecule is not substantially inhibited;

Provide a polymerase;

The PCR product is detected to identify the double-stranded target sequence.

In yet another aspect, the invention relates to an oligonucleotide pair for detecting fish pathogens.

In yet another aspect, the present invention relates to an oligonucleotide pair and oligonucleotide probe for detecting fish pathogens.

It should be further understood that in certain embodiments, the oligonucleotide pairs and / or oligonucleotide probes disclosed herein can be used for variations in various basic PCR techniques, such as, but not limited to, reverse transcription polymerization Enzyme chain reaction (RT-PCR), thermal convection polymerase chain reaction (cPCR), real-time polymerase chain reaction (real-time PCR), nested polymerase chain reaction (nested PCR), and thermal asymmetry Sex interleaved polymerase chain reaction (thermal asymmetric interlaced PCR, TAIL-PCR).

As used herein, the term "fish pathogen" means viral, bacterial, and parasitic pathogens found in and out of fish organisms. Examples of fish pathogens include, but are not limited to: Piscine Reovirus (PRV), Infectious Pancreatic Necrosis Virus (IPNV), Infectious Salmon Anemia Virus (ISAV) ), Piscine myocarditis virus (PMCV), Salmonid alphavirus (SAV), Neoparamoeba perurans, Candidatus Branchicomonas cysticola (CBc), Desmozoon lepeophtherii (Des) , Salmon Gill Poxvirus (SGPV), Salmon Rickettsia (Piscirickettsia salmonis), and Salmon Kidney Bacteria (Renibacterium salmoninarum).

As used herein, the term "thermal convection polymerase chain reaction (cPCR)" refers to a polymerase chain reaction in which the bottom of a tubular container containing a PCR sample is embedded in a stable heat source and the PCR is controlled The parameters, including the total volume, viscosity, surface temperature of the PCR sample, and the inner diameter of the tubular container, cause the temperature gradient from the bottom to the top of the PCR sample to decrease, induce thermal convection, and cause denaturation and adhesion of the PCR sample The polymerization occurs sequentially and repeatedly in different areas of the tubular container. For a detailed description of thermal convection polymerase chain reaction (cPCR), see, eg, US Patent No. 8,187,813, which is incorporated herein by reference in its entirety.

As used herein, the term "fluorescent molecule" means a substance or a portion thereof, which is capable of displaying fluorescence within a detectable range. As used herein, the term "fluorescence-inhibiting molecule" means a substance or a portion thereof, which is capable of inhibiting the fluorescence emitted by the fluorescent molecule when excited by a light source. In certain embodiments, the terms "fluorescent molecule" and "fluorescence inhibitory molecule" are fluorescent molecules and fluorescence inhibitory molecules of the TaqMan ^™ analysis kit (Applied Biosystems Inc., California, United States). For a detailed description of the TaqMan ^TM analysis kit, please see, for example, Holland et al., Proc. Natl. Acad. Sci, USA (1991) 88: 7276-7280; US Patent Nos. 5,538,848, 5,723,591, 5,876,930, and 7,413,708 are all cited. Way to incorporate its entirety into this article.

Examples of the fluorescent molecule include, but are not limited to, 3- (ε-carboxy) -3'-ethyl-5,5'-dimethylhexylcarbocyanine (3- (ε-carboxypentyl) -3'- ethyl-5,5'-dimethyloxa-carbocyanine (CYA), 6-carboxyfluorescein (FAM), 5,6-carboxyrhodamine-L LO (5,6-carboxyrhodamine-lO, R110), 6-carboxyrhodamine-6G (6-carboxyrhodamine-6G, R6G), N ', N', N ', N'-tetramethyl-6-carboxyrhodamine (N', N ', N', N'- tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 2 ', 4', 5 ', 7'-tetrachloro 4-7-dichloro fluorescence (2 ', 4', 5 ', 7'-tetrachloro-4-7-dichlorofluorescein, TET), 2', 7-dimethoxy-4 ', 5'-6 carboxyrhodamine (2', 7 -dimethoxy-4 ', 5'-6carboxyrhodamine, JOE), 6-carboxy-2', 4,4 ', 5', 7,7'-hexachlorofluorescein (6-carboxy-2 ', 4,4' , 5 ', 7,7'-hexachlorofluorescein, HEX), ALEXA fluorescence, Cy3 fluorescence and Cy5 fluorescence. Examples of the fluorescence inhibitory molecules include, but are not limited to, 4- (4'-dimethylamino-phenylazo) -benzoic acid (4- (4'-dimethylamino-phenylazo) -benzoic acid, Dabcyl), black holes Fluorescence inhibitor 1 (Black HoleQuencher1, BHQ1), black hole fluorescence inhibitor 2 (Black HoleQuencher2, BHQ2), black hole fluorescence inhibitor 3 (Black HoleQuencher3, BHQ3) Ditch conjugate (dihydro cyclopyrolo indole tripeptide mineral groove binder, MGB), tetramethylrhodamine (tetramethylrhodamine, TAMRA). In certain embodiments, the fluorescent molecule is 6-carboxyfluorescein (FAM), and the fluorescence inhibitory molecule is dihydrocyclopyrroloindole tripeptide minor groove conjugate (MGB). In certain embodiments, the fluorescent molecule is 6-carboxyfluorescein (FAM), and the fluorescence inhibitory molecule is black hole fluorescence inhibitor 1 (BHQ1) or dihydrocyclopyrroloindole tripeptide minor groove conjugate (MGB).

Unless otherwise defined herein, the scientific and technical terms used in conjunction with this document shall have the meaning commonly understood by those of ordinary skill in the art. In addition, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present invention can generally be carried out according to conventional methods known in the art. Generally speaking, the nomenclature described in this article to link the following technologies, as well as the technologies of biochemistry, enzymology, molecular and cell biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization are all in the field Known and frequent users. Unless otherwise stated, the methods and techniques of the present invention may generally be performed according to conventional methods known in the art, and are described in various general and more specific references that are cited and discussed in this specification.

The invention is further illustrated by the following examples, which should not be interpreted as a further limitation in any way. The entire contents of all the cited documents cited in this application (including references, approved patents, published patent applications, and patent applications filed together) are hereby expressly incorporated into this case by reference .

Examples

Example 1 Detection of pathogenic fish respiratory tract and enterovirus (PRV)

Pathogen fish respiratory tract and enterovirus (PRV) L1 segment gene (GenBank accession No. KY429943) and Sigma 3 protein gene (GenBank accession No. KX844958) fragments were inserted into a selection vector, the selection vector It can be, but not limited to, pUC57, pGEM-T to obtain pPRV-L1 and pPRV-S3 plasmids.

1. Traditional polymerase chain reaction

50 l PCR mixture was subjected to a conventional PCR used contains: 10 ⁶ copy number pPRV-L1 plasmid or pPRV-S3, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.2μM dNTP and 1.25U Taq DNA polymerase. Amplify the reaction in a thermal cycler (eg, but not limited to PC818, Astec Co. Ltd., Japan), and include an initial cycle of denaturation at 94 ° C for 3 minutes, and 35 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 30 seconds. The amplified product was then analyzed with 15% polyacrylamide gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) and visualized by staining with ethidium bromide.

The results of traditional PCR are shown in Table 1. Each primer pair amplified the correct size fragment of each target sequence, while no target sequence was amplified in the negative control group (results not shown). The results indicate that each primer pair can be used in traditional PCR amplification reactions to detect the presence of fish respiratory tract and enterovirus (PRV).

Table 1 Results of traditional PCR performed by combining each primer pair and probe

正向引物Forward primer	反向引物Reverse primer	合成片段大小(bp)Synthetic fragment size (bp)
PRV-F1(SEQ ID NO:1)PRV-F1 (SEQ ID NO: 1)	PRV-R1(SEQ ID NO:2)PRV-R1 (SEQ ID NO: 2)	7070
PRV-F2(SEQ ID NO:4)PRV-F2 (SEQ ID NO: 4)	PRV-R1(SEQ ID NO:2)PRV-R1 (SEQ ID NO: 2)	9292
PRV-F2(SEQ ID NO:4)PRV-F2 (SEQ ID NO: 4)	PRV-R2(SEQ ID NO:5)PRV-R2 (SEQ ID NO: 5)	9898
PRV-F3(SEQ ID NO:6)PRV-F3 (SEQ ID NO: 6)	PRV-R3(SEQ ID NO:7)PRV-R3 (SEQ ID NO: 7)	8383
PRV-F4(SEQ ID NO:9)PRV-F4 (SEQ ID NO: 9)	PRV-R4(SEQ ID NO:10)PRV-R4 (SEQ ID NO: 10)	7777
PRV-F5(SEQ ID NO:11)PRV-F5 (SEQ ID NO: 11)	PRV-R5(SEQ ID NO:12)PRV-R5 (SEQ ID NO: 12)	7676

2. Thermal convection polymerase chain reaction (cPCR)

50μl of PCR mix containing 5μl of Respiratory and enterovirus fish (the PRV) gene segments of the L1 gene or the Sigma 3 RNA (respectively ^102, ^103, ^104, ^105, ¹⁰⁶ the number of copies / μl) or pPRV-L1 or pPRV-S3 plasmids (10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ copies), 0.01-2 μM forward primer, 0.01-2 μM reverse primer, 0.01-2 μM probe ( The 5 'and 3' ends of each probe sequence are respectively joined with a fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescent inhibitor molecule black hole fluorescent inhibitor 1 (BHQ1)), 0.2 μM dNTP, 1X cPCR buffer, and 1-5U Taq DNA polymerase, 1-5U reverse transcriptase. The PCR mixture is added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) instrument for a specified period of time (approximately 30-45 minutes). The cPCR instrument was used to detect FAM fluorescence in each sample. The above cPCR analysis test was repeated 8 times (n = 8) to evaluate the sensitivity of each estimated primer to the probe.

The results of the sensitivity tests shown in Table 2, combinations of primer pair and a probe to detect Jieke 100% correct sample number contained ¹⁰² copies / μl of Respiratory and enterovirus fish (the PRV) L1 of segments Gene or Sigma 3 protein gene RNA or 10 ² copy number pPRV-L1 or pPRV-S3 plasmid, its sensitivity can reach 10 ² copy number / μl and 10 ² copy number.

Table 2 Sensitivity test results of each primer pair and probe combination (n = 8)

In addition, different fish pathogens plasmid (the copy number of ¹⁰⁶ / μl) as a template cPCR analysis of each specific primer pair and probe combination. The cPCR method is as described above. The results of the specificity test are shown in Table 3. Each primer pair and probe combination can correctly detect samples containing fish respiratory tract and enterovirus (PRV), but no infectious pancreatic necrosis can be detected. Viruses (IPNV), Infectious Salmon Anemia Virus (ISAV), Fish Myocarditis Virus (PMCV), Salmon A Virus (SAV), Peruran New Para amoeba (N. perurans, indicated by AGD in the table), Candidatus Branchiomonas cysticola (CBc), microspore insect Desmozoon lepeophtherii (Des), salmon parrot virus (SGPV), salmon rickettsia (P. salmononis, represented by SRS in the table), and salmon kidney bacteria (R. salmoninarum, in BKD in the table. The results show that each primer pair and probe combination are specific.

Table 3 Specific test results of each primer pair and probe combination

"+" Indicates that a fluorescent signal was detected in the sample, and "-" indicates that no fluorescent signal was detected in the sample.

3. Real-time PCR (qPCR)

Use diluted pPRV-L1 or pPRV-S3 plasmids (10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ copy numbers, respectively) in a real-time PCR instrument (for example, but not limited to ABI StepOnePlus ^™ ; Applied BioSystem, Life Technologies, California, USA) for real-time PCR analysis. Contain a fluorescent molecule 6 with 2μl pPRV-L1 or pPRV-S3 plasmid, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.01-2μM probe (5 'and 3' ends of each probe sequence respectively) -Carboxy fluorescein (FAM) and a fluorescence inhibitor molecule black hole fluorescence inhibitor 1 (BHQ1)), a commercial RT-PCR kit with a total volume of 20 μl (for example, but not limited to, OneStepPrimeScript ^™ RT-PCR Kit; Takara Bio Inc ., Japan) for real-time PCR analysis. The real-time PCR program is 42 ° C for 5 minutes, 94 ° C for 10 seconds, and 40 cycles of 94 ° C for 10 seconds and 60 ° C for 30 minutes. Record the results of the fluorescence measurement in the 60 ° C step and calculate. Standard curve for real-time PCR analysis of serially diluted (10-fold) pPRV-L1 or pPRV-S3 plasmids. The results showed that at least 10 ² copy number pPRV-L1 or pPRV-S3 plasmid can be detected, and each primer pair R ² value and the standard probe combinations curves are greater than 0.99, it indicates the primer of the present invention on the Probes can be used for real-time PCR and produce believable results.

The results of the above examples show that the primer pair and the probe of the present invention can be used in the cPCR amplification reaction to detect the presence of fish respiratory tract and enterovirus (PRV), and have high sensitivity and specificity.

Example 2 Detection of pathogenic infectious pancreatic necrosis virus (IPNV)

The fragments of the pathogen infectious pancreatic necrosis virus (IPNV) VP5 and polyprotein gene (GenBank accession No. KY548514) are inserted into a selection vector, which can be, but is not limited to, pUC57 , PGEM-T, to get pIPNV plasmid.

1. Traditional polymerase chain reaction

50 l PCR mixture was subjected to a conventional PCR used contained: pIPNV ¹⁰⁶ plasmid copy number, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.2μM dNTP and 1.25U Taq DNA polymerase. Amplify the reaction in a thermal cycler (eg, but not limited to PC818, Astec Co. Ltd., Japan), and include an initial cycle of denaturation at 94 ° C for 3 minutes, and 35 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 30 seconds. The amplified product was then analyzed with a 15% polyacrylamide gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) and visualized by ethidium bromide staining.

The results of traditional PCR are shown in Table 4. Each primer pair amplified the correct size fragment of each target sequence, while no target sequence was amplified in the negative control group (results not shown). The results indicate that each primer pair can be used in traditional PCR amplification reactions to detect the presence of infectious pancreatic necrosis virus (IPNV).

Table 4 Results of traditional PCR performed with each primer pair and probe

正向引物Forward primer	反向引物Reverse primer	合成片段大小(bp)Synthetic fragment size (bp)
IPNV-F1(SEQ ID NO:14)IPNV-F1 (SEQ ID NO: 14)	IPNV-R1(SEQ ID NO:15)IPNV-R1 (SEQ ID NO: 15)	8686
IPNV-F2(SEQ ID NO:17)IPNV-F2 (SEQ ID NO: 17)	IPNV-R2(SEQ ID NO:18)IPNV-R2 (SEQ ID NO: 18)	104104
IPNV-F3(SEQ ID NO:20)IPNV-F3 (SEQ ID NO: 20)	IPNV-R3(SEQ ID NO:21)IPNV-R3 (SEQ ID NO: 21)	8888

正向引物Forward primer	反向引物Reverse primer	合成片段大小(bp)Synthetic fragment size (bp)
IPNV-F4(SEQ ID NO:23)IPNV-F4 (SEQ ID NO: 23)	IPNV-R4(SEQ ID NO:24)IPNV-R4 (SEQ ID NO: 24)	9191

2. Thermal convection polymerase chain reaction (cPCR)

Multimeric protein VP5 gene RNA PCR mixture containing 5μl 50μl of infectious pancreatic necrosis virus (of IPNV) (respectively ^102, ^103, ^104, ^105, ¹⁰⁶ the number of copies / μl) or pIPNV plasmid ( 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ copies), 0.01-2 μM forward primer, 0.01-2 μM reverse primer, 0.01-2 μM probe (the 5 ′ end of each probe sequence and At the 3 'end, a fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescent inhibitory molecule black hole fluorescent inhibitor 1 (BHQ1)), 0.2 μM dNTP, 1X cPCR buffer, and 1-5U Taq DNA polymerase, 1 -5U reverse transcriptase. The PCR mixture is added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) instrument for a specified period of time (approximately 30-45 minutes). The cPCR instrument was used to detect FAM fluorescence in each sample. The above cPCR analysis test was repeated 8 times (n = 8) to evaluate the sensitivity of each estimated primer to the probe.

The combination of the sensitivity of the test results shown in Table 5, each of the primer pairs and probes Jieke 100% correct detected contained in the sample number 10 ² copies / μl infectious pancreatic necrosis virus (of IPNV) and the plurality of VP5 The RNA of the polyprotein gene or the 10 ² copy number pIPNV plasmid has a sensitivity of up to 10 ² copy number / μl and 10 ² copy number.

Table 5 Sensitivity test results of each primer pair and probe combination (n = 8)

In addition, different fish pathogens plasmid (the copy number of ¹⁰⁶ / μl) as a template cPCR analysis of each specific primer pair and probe combination. The cPCR method is as described above. The results of the specificity test are shown in Table 6. Each primer pair and probe combination can correctly detect samples containing infectious pancreatic necrosis virus (IPNV), but no fish respiratory tract and intestinal tract. Viruses (PRV), Infectious Salmon Anemia Virus (ISAV), Fish Myocarditis Virus (PMCV), Salmon A Virus (SAV), Perulan New Paraproteans (N. perurans, indicated by AGD in the table), Candidatus Branchiomonas cysticola (CBc), microspore insect Desmozoon lepeophtherii (Des), salmon parrot virus (SGPV), salmon rickettsia (P. salmononis, represented by SRS in the table), and salmon kidney bacteria (R. salmoninarum, in BKD in the table. The results show that each primer pair and probe combination are specific.

Table 6 Specific test results of each primer pair and probe combination

3. Real-time PCR (qPCR)

Use diluted pIPNV plasmids (10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ copy numbers) on a real-time PCR instrument (for example, but not limited to ABI StepOnePlus ^TM ; Applied BioSystem, Life Technologies, California, USA) real-time PCR analysis. Contain a fluorescent molecule 6-carboxyfluorescein (FAM) with 2μl pIPNV plasmid, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.01-2μM probe (5 'and 3' ends of each probe sequence respectively) ) And a fluorescent inhibitor black hole fluorescent inhibitor 1 (BHQ1)), a commercial RT-PCR kit with a total volume of 20 μl (eg, but not limited to, OneStep PrimeScript ^™ RT-PCR Kit; Takara Bio Inc., Japan) Real-time PCR analysis. The real-time PCR program is 42 ° C for 5 minutes, 94 ° C for 10 seconds, and 40 cycles of 94 ° C for 10 seconds and 60 ° C for 30 minutes. Record the results of the fluorescence measurement in the 60 ° C step and calculate. Standard curve for real-time PCR analysis of serially diluted (10-fold) pIPNV plasmids. The results show that at least 10 copies of the pIPNV plasmid can be detected, and the R ² value of the standard curve of each primer pair and probe combination is greater than 0.99, indicating that the primer pair and probe of the present invention can be used for real-time PCR and produce credible results.

The results of the above examples show that the primer pair and probe of the present invention can be used in the cPCR amplification reaction to detect the presence of infectious pancreatic necrosis virus (IPNV), and have high sensitivity and specificity.

Example 3 Detection of pathogen infectious salmon anemia virus (ISAV)

The fragments of the open reading frame (ORF) 1 and ORF2 genes (GenBank accession No. KX424589) of pathogen infectious salmon anemia virus (ISAV) are inserted into a selection vector, which can be, but is not limited to, pUC57, pGEM-T to get pISAV plasmid.

1. Traditional polymerase chain reaction

50 l PCR mixture was subjected to a conventional PCR used contained: pISAV ¹⁰⁶ plasmid copy number, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.2μM dNTP and 1.25U Taq DNA polymerase. Amplify the reaction in a thermal cycler (eg, but not limited to PC818, Astec Co. Ltd., Japan), and include an initial cycle of denaturation at 94 ° C for 3 minutes, and 35 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 30 seconds. The amplified product was then analyzed with a 15% polyacrylamide gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) and visualized by ethidium bromide staining.

The results of conventional PCR are shown in Table 7. Each primer pair amplified the correct size fragment of each target sequence, while no target sequence was amplified in the negative control group (results not shown). The results indicate that each primer pair can be used in traditional PCR amplification reactions to detect the presence of infectious salmon anemia virus (ISAV).

Table 7 Results of traditional PCR performed by combining each primer pair and probe

正向引物Forward primer	反向引物Reverse primer	合成片段大小(bp)Synthetic fragment size (bp)
ISAV-F1(SEQ ID NO:26)ISAV-F1 (SEQ ID NO: 26)	ISAV-R1(SEQ ID NO:27)ISAV-R1 (SEQ ID NO: 27)	8787
ISAV-F2(SEQ ID NO:29)ISAV-F2 (SEQ ID NO: 29)	ISAV-R2(SEQ ID NO:30)ISAV-R2 (SEQ ID NO: 30)	8383
ISAV-F3(SEQ ID NO:32)ISAV-F3 (SEQ ID NO: 32)	ISAV-R3(SEQ ID NO:33)ISAV-R3 (SEQ ID NO: 33)	7777
ISAV-F4(SEQ ID NO:35)ISAV-F4 (SEQ ID NO: 35)	ISAV-R4(SEQ ID NO:36)ISAV-R4 (SEQ ID NO: 36)	8383
ISAV-F5(SEQ ID NO:37)ISAV-F5 (SEQ ID NO: 37)	ISAV-R5(SEQ ID NO:38)ISAV-R5 (SEQ ID NO: 38)	9696

2. Thermal convection polymerase chain reaction (cPCR)

ORF1 and RNA ORF2 gene PCR mixture 50μl containing 5μl infectious salmon anemia virus (ISAV) (respectively ^102, ^103, ^104, ^105, ¹⁰⁶ the number of copies / μl) or pISAV plasmids (respectively 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ copies), 0.01-2 μM forward primer, 0.01-2 μM reverse primer, 0.01-2 μM probe (5 ′ end and 3 ′ end of each probe sequence Join a fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescent inhibitor black hole fluorescent inhibitor 1 (BHQ1)), 0.2μM dNTP, 1X cPCR buffer, and 1-5U Taq DNA polymerase, 1-5U reverse Transcriptase. The PCR mixture is added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) instrument for a specified period of time (approximately 30-45 minutes). The cPCR instrument was used to detect FAM fluorescence in each sample. The above cPCR analysis test was repeated 8 times (n = 8) to evaluate the sensitivity of each estimated primer to the probe.

The results of the sensitivity tests shown in Table 8, and the combination of primer pairs Jieke 100% correct probe detects ORF1 and ORF2 genes contained in the sample number 10 ² copies / μl of salmon infectious anemia virus (ISAV) of RNA or 10 ² copy number pISAV plasmid, its sensitivity can reach 10 ² copy number / μl and 10 ² copy number.

Table 8 Sensitivity test results of each primer pair and probe combination (n = 8)

In addition, different fish pathogens plasmid (the copy number of ¹⁰⁶ / μl) as a template cPCR analysis of each specific primer pair and probe combination. The cPCR method is as described above. The results of the specificity test are shown in Table 9. Each primer pair and probe combination can correctly detect samples containing infectious salmon anemia virus (ISAV), but no infectious pancreatic necrosis virus (IPNV), Fish Respiratory and Enteroviruses (PRV), Fish Myocarditis Virus (PMCV), Salmon Alpha Virus (SAV), Perulans New Paramoans (N. perurans, indicated by AGD in the table), Candidatus Branchiomonas cysticola (CBc), microspore insect Desmozoon lepeophtherii (Des), salmon parrot virus (SGPV), salmon rickettsia (P. salmononis, represented by SRS in the table), and salmon kidney bacteria (R. salmoninarum, in BKD in the table. The results show that each primer pair and probe combination are specific.

Table 9 Specific test results of each primer pair and probe combination

3. Real-time PCR (qPCR)

Use diluted pISAV plasmids (10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ copy numbers) on a real-time PCR machine (for example, but not limited to ABI StepOnePlus ^TM ; Applied BioSystem, Life Technologies, California, USA) real-time PCR analysis. Contain a fluorescent molecule 6-carboxyfluorescein (FAM) with 2μl pISAV plasmid, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.01-2μM probe (5 'and 3' ends of each probe sequence respectively) ) And a fluorescent inhibitor black hole fluorescent inhibitor 1 (BHQ1)), a commercial RT-PCR kit with a total volume of 20 μl (eg, but not limited to, OneStep PrimeScript ^™ RT-PCR Kit; Takara Bio Inc., Japan) Real-time PCR analysis. The real-time PCR program is 42 ° C for 5 minutes, 94 ° C for 10 seconds, and 40 cycles of 94 ° C for 10 seconds and 60 ° C for 30 minutes. Record the results of the fluorescence measurement in the 60 ° C step and calculate. Standard curve for real-time PCR analysis of serially diluted (10-fold) pISAV plasmids. The results show, pISAV least ¹⁰² plasmid copy number can be detected, and each primer pair ² values to a standard curve of probe combinations are greater than 0.99 R, represents a primer with a probe of the present invention may be used Real-time PCR and produce credible results.

The results of the above examples show that the primer pair and probe of the present invention can be used in the cPCR amplification reaction to detect the presence of infectious salmon anemia virus (ISAV), and have high sensitivity and specificity.

Example 4 Detection of pathogen fish myocarditis virus (PMCV)

A fragment of the genome (GenBank accession No. HQ339954) of the pathogen fish myocarditis virus (PMCV) is inserted into a selection vector, which may be, but not limited to, pUC57, pGEM-T to obtain the pPMCV plasmid.

1. Traditional polymerase chain reaction

50 l PCR mixture was subjected to a conventional PCR used contained: pPMCV ¹⁰⁶ plasmid copy number, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.2μM dNTP and 1.25U Taq DNA polymerase. Amplify the reaction in a thermal cycler (eg, but not limited to PC818, Astec Co. Ltd., Japan), and include an initial cycle of denaturation at 94 ° C for 3 minutes, and 35 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 30 seconds. The amplified product was then analyzed with a 15% polyacrylamide gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) and visualized by ethidium bromide staining.

The results of conventional PCR are shown in Table 10. Each primer pair amplified the correct size fragment of each target sequence, while no target sequence was amplified in the negative control group (results not shown). The results indicate that each primer pair can be used in traditional PCR amplification reactions to detect the presence of fish myocarditis virus (PMCV).

Table 10 Results of traditional PCR performed by combining each primer pair and probe

正向引物Forward primer	反向引物Reverse primer	合成片段大小(bp)Synthetic fragment size (bp)
PMCV-F1(SEQ ID NO:39)PMCV-F1 (SEQ ID NO: 39)	PMCV-R1(SEQ ID NO:40)PMCV-R1 (SEQ ID NO: 40)	8181
PMCV-F2(SEQ ID NO:42)PMCV-F2 (SEQ ID NO: 42)	PMCV-R2(SEQ ID NO:43)PMCV-R2 (SEQ ID NO: 43)	8181
PMCV-F3(SEQ ID NO:45)PMCV-F3 (SEQ ID NO: 45)	PMCV-R3(SEQ ID NO:46)PMCV-R3 (SEQ ID NO: 46)	9595
PMCV-F4(SEQ ID NO:48)PMCV-F4 (SEQ ID NO: 48)	PMCV-R4(SEQ ID NO:49)PMCV-R4 (SEQ ID NO: 49)	8080
PMCV-F5(SEQ ID NO:51)PMCV-F5 (SEQ ID NO: 51)	PMCV-R4(SEQ ID NO:49)PMCV-R4 (SEQ ID NO: 49)	100100
PMCV-F6(SEQ ID NO:52)PMCV-F6 (SEQ ID NO: 52)	PMCV-R4(SEQ ID NO:49)PMCV-R4 (SEQ ID NO: 49)	8383

2. Thermal convection polymerase chain reaction (cPCR)

50 μl of the PCR mixture contains 5 μl of the RNA of the genome fragment of the fish myocarditis virus (PMCV) (10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ copy number / μl) or the pPMCV plasmid (10 ² , 10 ³ respectively) , 10 ⁴ , 10 ⁵ , 10 ⁶ copy numbers), 0.01-2 μM forward primer, 0.01-2 μM reverse primer, 0.01-2 μM probe (each probe sequence 5 'end and 3' end were joined with a fluorescence The molecule 6-carboxyfluorescein (FAM) and a fluorescence-inhibiting molecule black hole fluorescence inhibitor 1 (BHQ1)), 0.2 μM dNTP, 1X cPCR buffer, and 1-5U Taq DNA polymerase, 1-5U reverse transcriptase. The PCR mixture is added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) instrument for a specified period of time (approximately 30-45 minutes). The cPCR instrument was used to detect FAM fluorescence in each sample. The above cPCR analysis test was repeated 8 times (n = 8) to evaluate the sensitivity of each estimated primer to the probe.

The results of the sensitivity test are shown in Table 11. The combination of each primer pair and probe can 100% correctly detect the RNA or 10 of the genome fragment containing 10 ² copies / μl of the fish myocarditis virus (PMCV) in the sample. ² pPMCV plasmid copy number, with a sensitivity of up to ¹⁰² the number of copies / μl, and ¹⁰² copy numbers.

Table 11 Sensitivity test results of each primer pair with probe (n = 8)

In addition, different fish pathogens plasmid (the copy number of ¹⁰⁶ / μl) as a template cPCR analysis of each specific primer pair and probe combination. The cPCR method is as described above. The results of the specificity test are shown in Table 12. Each primer pair and probe combination can correctly detect samples containing fish myocarditis virus (PMCV), but no fish respiratory and enterovirus (PRV) ), Infectious Pancreatic Necrosis Virus (IPNV), Infectious Salmon Anemia Virus (ISAV), Salmon Alpha Virus (SAV), Perulan New Paramotes (N. perurans, indicated by AGD in the table), Candidatus Branchiomonas cysticola (CBc), microspore insect Desmozoon lepeophtherii (Des), salmon parrot virus (SGPV), salmon rickettsia (P. salmononis, represented by SRS in the table), and salmon kidney bacteria (R. salmoninarum, in BKD in the table. The results show that each primer pair and probe combination are specific.

Table 12 Specific test results of each primer pair and probe combination

3. Real-time PCR (qPCR)

Use diluted pPMCV plasmids (10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ copies) on a real-time PCR machine (for example, but not limited to ABI StepOnePlus ^TM ; Applied BioSystem, Life Technologies, California, USA) real-time PCR analysis. Contain a fluorescent molecule 6-carboxyfluorescein (FAM) with 2μl pPMCV plasmid, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.01-2μM probe (5 'end and 3' end of each probe sequence respectively) ) And a fluorescent inhibitor black hole fluorescent inhibitor 1 (BHQ1)), a commercial RT-PCR kit with a total volume of 20 μl (eg, but not limited to, OneStep PrimeScript ^™ RT-PCR Kit; Takara Bio Inc., Japan) Real-time PCR analysis. The real-time PCR program is 42 ° C for 5 minutes, 94 ° C for 10 seconds, and 40 cycles of 94 ° C for 10 seconds and 60 ° C for 30 minutes. Record the results of the fluorescence measurement in the 60 ° C step and calculate. Standard curve for real-time PCR analysis of serially diluted (10-fold) pPMCV plasmids. The results show, pPMCV least ¹⁰² plasmid copy number can be detected, and each primer pair ² values to a standard curve of probe combinations are greater than 0.99 R, represents a primer with a probe of the present invention may be used Real-time PCR and produce credible results.

The results of the above examples show that the primer pair and probe of the present invention can be used in the cPCR amplification reaction to detect the presence of fish myocarditis virus (PMCV), and have high sensitivity and specificity.

Example 5 Detection of Pathogen Salmon Alpha Virus (SAV)

A fragment of the genome (GenBank accession No. KC122926) of the pathogen Salmon Alpha Virus (SAV) is inserted into a selection vector, which may be, but not limited to, pUC57, pGEM-T, to obtain the pSAV plasmid.

1. Traditional polymerase chain reaction

50 l PCR mixture was subjected to a conventional PCR used contained: pSAV ¹⁰⁶ plasmid copy number, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.2μM dNTP and 1.25U Taq DNA polymerase. Amplify the reaction in a thermal cycler (eg, but not limited to PC818, Astec Co. Ltd., Japan), and include an initial cycle of denaturation at 94 ° C for 3 minutes, and 35 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 30 seconds. The amplified product was then analyzed with a 15% polyacrylamide gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) and visualized by ethidium bromide staining.

The results of conventional PCR are shown in Table 13. Each primer pair amplified the correct size fragment of each target sequence, while no target sequence was amplified in the negative control group (results not shown). The results indicate that each primer pair can be used in a traditional PCR amplification reaction to detect the presence of salmonella virus (SAV).

Table 13 Results of traditional PCR performed by combining each primer pair and probe

正向引物Forward primer	反向引物Reverse primer	合成片段大小(bp)Synthetic fragment size (bp)
SAV-F1(SEQ ID NO:53)SAV-F1 (SEQ ID NO: 53)	SAV-R1(SEQ ID NO:54)SAV-R1 (SEQ ID NO: 54)	8787
SAV-F2(SEQ ID NO:56)SAV-F2 (SEQ ID NO: 56)	SAV-R2(SEQ ID NO:57)SAV-R2 (SEQ ID NO: 57)	7777
SAV-F3(SEQ ID NO:59)SAV-F3 (SEQ ID NO: 59)	SAV-R3(SEQ ID NO:60)SAV-R3 (SEQ ID NO: 60)	110110
SAV-F4(SEQ ID NO:62)SAV-F4 (SEQ ID NO: 62)	SAV-R4(SEQ ID NO:63)SAV-R4 (SEQ ID NO: 63)	8484
SAV-F5(SEQ ID NO:65)SAV-F5 (SEQ ID NO: 65)	SAV-R5(SEQ ID NO:66)SAV-R5 (SEQ ID NO: 66)	8383

2. Thermal convection polymerase chain reaction (cPCR)

RNA genome fragments PCR mixture 50μl containing 5μl salmon alphavirus (SAV) (respectively ^102, ^103, ^104, ^105, ¹⁰⁶ the number of copies / μl) or pSAV plasmids (respectively ^102, ¹⁰³ , 10 ⁴ , 10 ⁵ , 10 ⁶ copy numbers), 0.01-2 μM forward primer, 0.01-2 μM reverse primer, 0.01-2 μM probe (each probe sequence 5 'end and 3' end were joined with a fluorescence The molecule 6-carboxyfluorescein (FAM) and a fluorescence-inhibiting molecule black hole fluorescence inhibitor 1 (BHQ1)), 0.2 μM dNTP, 1X cPCR buffer, and 1-5U Taq DNA polymerase, 1-5U reverse transcriptase. The PCR mixture is added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) instrument for a specified period of time (approximately 30-45 minutes). The cPCR instrument was used to detect FAM fluorescence in each sample. The above cPCR analysis test was repeated 8 times (n = 8) to evaluate the sensitivity of each estimated primer to the probe.

The results of the sensitivity test are shown in Table 14. The combination of each primer pair and probe can 100% correctly detect the RNA or 10 of the genomic fragment containing 10 ² copies / μl of salmonella virus (SAV) in the sample. ² pSAV plasmid copy number, with a sensitivity of up to ¹⁰² the number of copies / μl, and ¹⁰² copy numbers.

Table 14 Sensitivity test results of each primer pair and probe combination (n = 8)

In addition, different fish pathogens plasmid (the copy number of ¹⁰⁶ / μl) as a template cPCR analysis of each specific primer pair and probe combination. The cPCR method is as described above. The results of the specificity test are shown in Table 15. The combination of each primer pair and probe can correctly detect the sample containing salmon alphavirus (SAV), but cannot detect the fish respiratory and enterovirus (PRV). ), Infectious Pancreatic Necrosis Virus (IPNV), Infectious Salmon Anemia Virus (ISAV), Fish Myocarditis Virus (PMCV), Perulan New Paraproteans (N. perurans, indicated by AGD in the table), Candidatus Branchiomonas cysticola (CBc), microspore insect Desmozoon lepeophtherii (Des), salmon parrot virus (SGPV), salmon rickettsia (P. salmononis, represented by SRS in the table), and salmon kidney bacteria (R. salmoninarum, in BKD in the table. . The results show that each primer pair and probe combination are specific.

Table 15 Specific test results of each primer pair and probe combination

3. Real-time PCR (qPCR)

Use diluted pSAV plasmids (10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ copy numbers) on a real-time PCR instrument (for example, but not limited to ABI StepOnePlus ^TM ; Applied BioSystem, Life Technologies, California, USA) real-time PCR analysis. Contain a fluorescent molecule 6-carboxyfluorescein (FAM) with 2μl pSAV plasmid, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.01-2μM probe (5 'end and 3' end of each probe sequence respectively) ) And a fluorescent inhibitor black hole fluorescent inhibitor 1 (BHQ1)), a commercial RT-PCR kit with a total volume of 20 μl (eg, but not limited to, OneStep PrimeScript ^™ RT-PCR Kit; Takara Bio Inc., Japan) Real-time PCR analysis. The real-time PCR program is 42 ° C for 5 minutes, 94 ° C for 10 seconds, and 40 cycles of 94 ° C for 10 seconds and 60 ° C for 30 minutes. Record the results of the fluorescence measurement in the 60 ° C step and calculate. Standard curve for real-time PCR analysis of serially diluted (10-fold) pSAV plasmids. The results show that at least 10 ² copy number pSAV plasmids can be detected, and the R ² value of the standard curve of each primer pair and probe combination is greater than 0.99, indicating that the primer pair and probe of the present invention can be used Real-time PCR and produce credible results.

The results of the above examples show that the primer pair and the probe of the present invention can be used in the cPCR amplification reaction to detect the presence of salmonella virus (SAV), and have high sensitivity and specificity.

Example 6 Detection of the pathogen Perulans new paraproteans (N. perurans)

N. perurans is the pathogen that causes Amoebic Mill Disease (AGD). The fragment of the 18S ribosomal ribonucleic acid (rRNA) gene (GenBank accession No. KU985058) of N. perurans of Perulan is inserted into a selection vector, which may be, but not limited , PUC57, pGEM-T, to obtain pAGD plasmid.

1. Traditional polymerase chain reaction

50 l PCR mixture was subjected to a conventional PCR used contained: pAGD ¹⁰⁶ plasmid copy number, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.2μM dNTP and 1.25U Taq DNA polymerase. Amplify the reaction in a thermal cycler (eg, but not limited to PC818, Astec Co. Ltd., Japan), and include an initial cycle of denaturation at 94 ° C for 3 minutes, and 35 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 30 seconds. The amplified product was then analyzed with a 15% polyacrylamide gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) and visualized by ethidium bromide staining.

The results of traditional PCR are shown in Table 16. Each primer pair amplified the correct size fragment of each target sequence, while no target sequence was amplified in the negative control group (results not shown). The results indicated that each primer pair can be used in traditional PCR amplification reactions to detect the presence of N. perurans.

Table 16 Results of traditional PCR performed by combining each primer pair and probe

正向引物Forward primer	反向引物Reverse primer	合成片段大小(bp)Synthetic fragment size (bp)
AGD-F1(SEQ ID NO:68)AGD-F1 (SEQ ID NO: 68)	AGD-R1(SEQ ID NO:69)AGD-R1 (SEQ ID NO: 69)	111111
AGD-F2(SEQ ID NO:71)AGD-F2 (SEQ ID NO: 71)	AGD-R2(SEQ ID NO:72)AGD-R2 (SEQ ID NO: 72)	9595

正向引物Forward primer	反向引物Reverse primer	合成片段大小(bp)Synthetic fragment size (bp)
AGD-F3(SEQ ID NO:74)AGD-F3 (SEQ ID NO: 74)	AGD-R3(SEQ ID NO:75)AGD-R3 (SEQ ID NO: 75)	8989
AGD-F4(SEQ ID NO:77)AGD-F4 (SEQ ID NO: 77)	AGD-R4(SEQ ID NO:78)AGD-R4 (SEQ ID NO: 78)	7070

2. Thermal convection polymerase chain reaction (cPCR)

50 μl of the PCR mix contains 5 μl of the RNA of the 18S rRNA gene of N. perurans (10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ copy number / μl) or pAGD plasmid ( 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ copies), 0.01-2 μM forward primer, 0.01-2 μM reverse primer, 0.01-2 μM probe (the 5 ′ end of each probe sequence and At the 3 'end, a fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescent inhibitory molecule black hole fluorescent inhibitor 1 (BHQ1)), 0.2 μM dNTP, 1X cPCR buffer, and 1-5U Taq DNA polymerase, 1 -5U reverse transcriptase. The PCR mixture is added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) instrument for a specified period of time (approximately 30-45 minutes). The cPCR instrument was used to detect FAM fluorescence in each sample. The above cPCR analysis test was repeated 8 times (n = 8) to evaluate the sensitivity of each estimated primer to the probe.

The results of the sensitivity test are shown in Table 17, and the combination of each primer pair and probe can detect 100% of the samples containing 10 ² copies / μl of N. perurans. The sensitivity of 18S rRNA gene RNA or 10 ² copy number pAGD plasmids can reach 10 ² copy number / μl and 10 ² copy number.

Table 17 Sensitivity test results of each primer pair and probe combination (n = 8)

In addition, different fish pathogens plasmid (the copy number of ¹⁰⁶ / μl) as a template cPCR analysis of each specific primer pair and probe combination. The cPCR method is as described above. The results of the specificity test are shown in Table 18. The combination of each primer pair and probe can correctly detect the sample containing N. perurans (N. perurans, indicated by AGD in the table), and Cannot detect fish respiratory and enterovirus (PRV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia virus (ISAV), fish myocarditis virus (PMCV), salmonella virus (SAV), Candidatus Branchiomonas cysticola (CBc), microspore insect Desmozoon lepeophtherii (Des), salmon parrot virus (SGPV), salmon rickettsia (P. salmononis, represented by SRS in the table), and salmon kidney bacteria (R. salmoninarum, in BKD in the table. The results show that each primer pair and probe combination are specific.

Table 18 Specific test results of each primer pair and probe combination

3. Real-time PCR (qPCR)

Use diluted pAGD plasmids (10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ copy numbers) on a real-time PCR instrument (for example, but not limited to ABI StepOnePlus ^TM ; Applied BioSystem, Life Technologies, California, USA) real-time PCR analysis. Contain a fluorescent molecule 6-carboxyfluorescein (FAM) with 2μl pAGD plasmid, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.01-2μM probe (5 'end and 3' end of each probe sequence respectively) ) And a fluorescent inhibitor black hole fluorescent inhibitor 1 (BHQ1)), a commercial RT-PCR kit with a total volume of 20 μl (eg, but not limited to, OneStep PrimeScript ^™ RT-PCR Kit; Takara Bio Inc., Japan) Real-time PCR analysis. The real-time PCR program is 42 ° C for 5 minutes, 94 ° C for 10 seconds, and 40 cycles of 94 ° C for 10 seconds and 60 ° C for 30 minutes. Record the results of the fluorescence measurement in the 60 ° C step and calculate. Standard curve for real-time PCR analysis of serially diluted (10-fold) pAGD plasmids. The results show that at least 10 ² copy number pAGD plasmids can be detected, and the R ² value of the standard curve of each primer pair and probe combination is greater than 0.99, indicating that the primer pair and probe of the present invention can be used Real-time PCR and produce credible results.

The results of the above examples indicate that the primer pair and probe of the present invention can be used in a cPCR amplification reaction to detect the presence of N. perurans, and have high sensitivity and specificity.

Example 7 Detection of Ca.B.cysticola (CBc)

The 16S ribosomal ribonucleic acid (rRNA) gene (GenBank accession No. JQ723599) fragment of the ca. -T to obtain the pCBc plasmid.

1. Traditional polymerase chain reaction

50 l PCR mixture was subjected to a conventional PCR used contained: pCBc ¹⁰⁶ plasmid copy number, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.2μM dNTP and 1.25U Taq DNA polymerase. Amplify the reaction in a thermal cycler (eg, but not limited to PC818, Astec Co. Ltd., Japan), and include an initial cycle of denaturation at 94 ° C for 3 minutes, and 35 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 30 seconds. The amplified product was then analyzed with a 15% polyacrylamide gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) and visualized by ethidium bromide staining.

The results of conventional PCR are shown in Table 19. Each primer pair amplified the correct size fragment of each target sequence, while no target sequence was amplified in the negative control group (results not shown). The results indicate that each primer pair can be used in traditional PCR amplification reactions to detect the presence of Ca.B. cysticola (CBc).

Table 19 Results of traditional PCR performed by combining each primer pair and probe

正向引物Forward primer	反向引物Reverse primer	合成片段大小(bp)Synthetic fragment size (bp)
CBc-F1(SEQ ID NO:80)CBc-F1 (SEQ ID NO: 80)	CBc-R1(SEQ ID NO:81)CBc-R1 (SEQ ID NO: 81)	9898
CBc-F2(SEQ ID NO:83)CBc-F2 (SEQ ID NO: 83)	CBc-R2(SEQ ID NO:84)CBc-R2 (SEQ ID NO: 84)	7070

2. Thermal convection polymerase chain reaction (cPCR)

50μl of PCR mix containing 5μl RNA Ca.B.cysticola (CBc) of the 16S rRNA gene (respectively ^102, ^103, ^104, ^105, ¹⁰⁶ the number of copies / μl) or pCBc plasmids (respectively ¹⁰² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ copies), 0.01-2 μM forward primer, 0.01-2 μM reverse primer, 0.01-2 μM probe (the 5 ′ end and 3 ′ end of each probe sequence are Conjugation of a fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescent inhibitor molecule black hole fluorescence inhibitor 1 (BHQ1)), 0.2 μM dNTP, 1X cPCR buffer, and 1-5U Taq DNA polymerase, 1-5U inversion Record enzymes. The PCR mixture is added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) instrument for a specified period of time (approximately 30-45 minutes). The cPCR instrument was used to detect FAM fluorescence in each sample. The above cPCR analysis test was repeated 8 times (n = 8) to evaluate the sensitivity of each estimated primer to the probe.

The results of the sensitivity test are shown in Table 20. Each primer pair and probe combination can 100% correctly detect the 16S rRNA gene of Ca.B. cysticola (CBc) containing 10 ² copies / μl in the sample. The sensitivity of RNA or 10 ² copy number pCBc plasmids can reach 10 ² copy number / μl and 10 ² copy number.

Table 20 Sensitivity test results of each primer pair and probe combination (n = 8)

In addition, different fish pathogens plasmid (the copy number of ¹⁰⁶ / μl) as a template cPCR analysis of each specific primer pair and probe combination. The cPCR method is as described above. The results of the specificity test are shown in Table 21. Each primer pair and probe combination can correctly detect samples containing Candidatus Branchiomonas cysticola (CBc), but no fish respiratory tract and enterovirus (PRV) ), Infectious Pancreatic Necrosis Virus (IPNV), Infectious Salmon Anemia Virus (ISAV), Fish Myocarditis Virus (PMCV), Salmon A Virus (SAV), Peruran's New Paraproteans (N. (Denoted by AGD), Microsporidian Desmozoon lepeophtherii (Des), salmon parrot virus (SGPV), salmon rickettsia (P. salmonis, represented by SRS in the table), and salmon kidney fungus (R. salmoninarum, in BKD in the table. The results show that each primer pair and probe combination are specific.

[Table 21] Specific test results of each primer pair and probe combination

3. Real-time PCR (qPCR)

Use diluted pCBc plasmids (10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ copy numbers) on a real-time PCR instrument (for example, but not limited to ABI StepOnePlus ^TM ; Applied BioSystem, Life Technologies, California, USA) real-time PCR analysis. Containing a fluorescent molecule 6-carboxyfluorescein (FAM) with 2μl pCBc plasmid, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.01-2μM probe (5 'end and 3' end of each probe sequence respectively) ) And a fluorescent inhibitor black hole fluorescent inhibitor 1 (BHQ1)), a commercial RT-PCR kit with a total volume of 20 μl (eg, but not limited to, OneStep PrimeScript ^™ RT-PCR Kit; Takara Bio Inc., Japan) Real-time PCR analysis. The real-time PCR program is 42 ° C for 5 minutes, 94 ° C for 10 seconds, and 40 cycles of 94 ° C for 10 seconds and 60 ° C for 30 minutes. Record the results of the fluorescence measurement in the 60 ° C step and calculate. Standard curve for real-time PCR analysis of serially diluted (10-fold) pCBc plasmids. The results show that at least 10 ² copy number pCBc plasmids can be detected, and the R ² value of the standard curve of each primer pair and probe combination is greater than 0.99, indicating that the primer pair and probe of the present invention can be used Real-time PCR and produce credible results.

The results of the above examples show that the primer pair and probe of the present invention can be used in the cPCR amplification reaction to detect the presence of Ca.B. cysticola (CBc), and have high sensitivity and specificity.

Example 8 Detection of the pathogen Microsporidium D. lepeophtherii (Des)

The 16S ribosomal ribonucleic acid (rRNA) gene (GenBank accession No. JQ723599) fragment of the pathogen Microsporidium D. lepeophtherii (Des) is inserted into a selection vector, which may be, but not limited to, pUC57 pGEM-T to obtain the pDes plasmid.

1. Traditional polymerase chain reaction

50 l PCR mixture was subjected to a conventional PCR used contained: pDes ¹⁰⁶ plasmid copy number, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.2μM dNTP and 1.25U Taq DNA polymerase. Amplify the reaction in a thermal cycler (eg, but not limited to PC818, Astec Co. Ltd., Japan), and include an initial cycle of denaturation at 94 ° C for 3 minutes, and 35 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 30 seconds. The amplified product was then analyzed with a 15% polyacrylamide gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) and visualized by ethidium bromide staining.

The results of traditional PCR are shown in Table 22. Each primer pair amplified the correct size fragment of each target sequence, while no target sequence was amplified in the negative control group (results not shown). The results indicate that each primer pair can be used in traditional PCR amplification reactions to detect the presence of D. lepeophtherii (Des).

Table 22 Results of traditional PCR performed by combining each primer pair and probe

正向引物Forward primer	反向引物Reverse primer	合成片段大小(bp)Synthetic fragment size (bp)
Des-F1(SEQ ID NO:86)Des-F1 (SEQ ID NO: 86)	Des-R1(SEQ ID NO:87)Des-R1 (SEQ ID NO: 87)	9292
Des-F2(SEQ ID NO:89)Des-F2 (SEQ ID NO: 89)	Des-R2(SEQ ID NO:90)Des-R2 (SEQ ID NO: 90)	8282
Des-F3(SEQ ID NO:92)Des-F3 (SEQ ID NO: 92)	Des-R3(SEQ ID NO:93)Des-R3 (SEQ ID NO: 93)	9191
Des-F4(SEQ ID NO:95)Des-F4 (SEQ ID NO: 95)	Des-R4(SEQ ID NO:97)Des-R4 (SEQ ID NO: 97)	8989
Des-F5(SEQ ID NO:96)Des-F5 (SEQ ID NO: 96)	Des-R4(SEQ ID NO:97)Des-R4 (SEQ ID NO: 97)	8787

2. Thermal convection polymerase chain reaction (cPCR)

RNA 16S rRNA gene PCR mix containing 5μl 50μl Nosema D.lepeophtherii (Des) (respectively ^102, ^103, ^104, ^105, ¹⁰⁶ the number of copies / μl) or pDes plasmids (respectively 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ copies), 0.01-2 μM forward primer, 0.01-2 μM reverse primer, 0.01-2 μM probe (5 ′ end and 3 ′ end of each probe sequence Join a fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescent inhibitor black hole fluorescent inhibitor 1 (BHQ1)), 0.2μM dNTP, 1X cPCR buffer, and 1-5U Taq DNA polymerase, 1-5U reverse Transcriptase. The PCR mixture is added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) instrument for a specified period of time (approximately 30-45 minutes). The cPCR instrument was used to detect FAM fluorescence in each sample. The above cPCR analysis test was repeated 8 times (n = 8) to evaluate the sensitivity of each estimated primer to the probe.

The results of the sensitivity test are shown in Table 23. Each primer pair and probe combination can 100% correctly detect the 16S rRNA gene containing 10 ² copies / μl of the microsporidian D. lepeophtherii (Des) in the sample. RNA or 10 ² copy number of pDes plasmid, its sensitivity can reach 10 ² copy number / μl and 10 ² copy number.

Table 23 Sensitivity test results of each primer pair and probe combination (n = 8)

In addition, different fish pathogens plasmid (the copy number of ¹⁰⁶ / μl) as a template cPCR analysis of each specific primer pair and probe combination. The cPCR method is as described above. The results of the specificity test are shown in Table 24. Each primer pair and probe combination can correctly detect the sample containing Desmozoon lepeophtherii (Des), but not the fish respiratory tract and enterovirus. (PRV), Infectious Pancreatic Necrosis Virus (IPNV), Infectious Salmon Anemia Virus (ISAV), Fish Myocarditis Virus (PMCV), Salmon Alpha Virus (SAV), and Perulan's New Paramotes (N.perurans, in (Denoted by AGD in the table), Candidatus Branchiomonas cysticola (CBc), salmon parrot virus (SGPV), salmon rickettsia (P. salmononis, represented by SRS in the table), and salmon kidney fungus (R. salmononirum, in BKD in the table. The results show that each primer pair and probe combination are specific.

Table 24 Specific test results of each primer pair and probe combination

3. Real-time PCR (qPCR)

Use diluted pDes plasmids (10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ copy numbers) on a real-time PCR instrument (for example, but not limited to ABI StepOnePlus ^TM ; Applied BioSystem, Life Technologies, California, USA) real-time PCR analysis. Containing a fluorescent molecule 6-carboxyfluorescein (FAM) with 2μl pDes plasmid, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.01-2μM probe (5 'end and 3' end of each probe sequence respectively) ) And a fluorescent inhibitor black hole fluorescent inhibitor 1 (BHQ1)), a commercial RT-PCR kit with a total volume of 20 μl (eg, but not limited to, OneStep PrimeScript ^™ RT-PCR Kit; Takara Bio Inc., Japan) Real-time PCR analysis. The real-time PCR program is 42 ° C for 5 minutes, 94 ° C for 10 seconds, and 40 cycles of 94 ° C for 10 seconds and 60 ° C for 30 minutes. Record the results of the fluorescence measurement in the 60 ° C step and calculate. Standard curve for real-time PCR analysis of serially diluted (10-fold) pDes plasmids. The results show that, at least ¹⁰² PDES plasmid copy number can be detected, and each primer pair ² values to a standard curve of probe combinations are greater than 0.99 R, represents a primer with a probe of the present invention may be used Real-time PCR and produce credible results.

The results of the above examples show that the primer pair and the probe of the present invention can be used in the cPCR amplification reaction to detect the presence of D. lepeophtherii (Des), and have high sensitivity and specificity.

Example 9 Detection of pathogen salmon parrot virus (SGPV)

The genome (GenBank accession No. KT159937) fragment of the pathogen salmon parrot virus (SGPV) is inserted into a selection vector, which may be, but not limited to, pUC57, pGEM-T, to obtain the pSGPV plasmid.

1. Traditional polymerase chain reaction

50 l PCR mixture was subjected to a conventional PCR used contained: pSGPV ¹⁰⁶ plasmid copy number, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.2μM dNTP and 1.25U Taq DNA polymerase. Amplify the reaction in a thermal cycler (eg, but not limited to PC818, Astec Co. Ltd., Japan), and include an initial cycle of denaturation at 94 ° C for 3 minutes, and 35 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 30 seconds. The amplified product was then analyzed with a 15% polyacrylamide gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) and visualized by ethidium bromide staining.

The results of conventional PCR are shown in Table 25. Each primer pair amplified the correct size fragment of each target sequence, while no target sequence was amplified in the negative control group (results not shown). The results indicate that each primer pair can be used in a traditional PCR amplification reaction to detect the presence of salmon parrot virus (SGPV).

Table 25 Results of traditional PCR performed by combining each primer pair and probe

正向引物Forward primer	反向引物Reverse primer	合成片段大小(bp)Synthetic fragment size (bp)
SGPV-F1(SEQ ID NO:99)SGPV-F1 (SEQ ID NO: 99)	SGPV-R1(SEQ ID NO:100)SGPV-R1 (SEQ ID NO: 100)	8484
SGPV-F2(SEQ ID NO:102)SGPV-F2 (SEQ ID NO: 102)	SGPV-R2(SEQ ID NO:103)SGPV-R2 (SEQ ID NO: 103)	7474
SGPV-F3(SEQ ID NO:105)SGPV-F3 (SEQ ID NO: 105)	SGPV-R3(SEQ ID NO:106)SGPV-R3 (SEQ ID NO: 106)	8282
SGPV-F4(SEQ ID NO:108)SGPV-F4 (SEQ ID NO: 108)	SGPV-R4(SEQ ID NO:109)SGPV-R4 (SEQ ID NO: 109)	106106

2. Thermal convection polymerase chain reaction (cPCR)

50 μl of the PCR mix contains 5 μl of the RNA of the genomic fragment of salmon parrot virus (SGPV) (10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ copy number / μl) or pSGPV plasmid (10 ² , 10 respectively) ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ copy numbers), 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.01-2μM probe (the 5 'end and the 3' end of each probe sequence are joined together Fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescent inhibitory molecule black hole fluorescence inhibitor 1 (BHQ1)), 0.2 μM dNTP, 1X cPCR buffer, and 1-5U Taq DNA polymerase, 1-5U reverse transcriptase . The PCR mixture is added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) instrument for a specified period of time (approximately 30-45 minutes). The cPCR instrument was used to detect FAM fluorescence in each sample. The above cPCR analysis test was repeated 8 times (n = 8) to evaluate the sensitivity of each estimated primer to the probe.

The results of the sensitivity test are shown in Table 26. Each primer pair and probe combination can detect 100% of the RNA or genomic fragment of salmon parvovirus (SGPV) containing 10 ² copies / μl in the sample. pSGPV plasmid copy number of ^102, a sensitivity of up to ¹⁰² the number of copies / μl, and ¹⁰² copy numbers.

Table 26 Sensitivity test results of each primer pair and probe combination (n = 8)

In addition, different fish pathogens plasmid (the copy number of ¹⁰⁶ / μl) as a template cPCR analysis of each specific primer pair and probe combination. The cPCR method is as described above. The results of the specificity test are shown in Table 27. Each primer pair and probe combination can correctly detect samples containing salmon parrot virus (SGPV), but no fish respiratory and enteroviruses ( PRV), Infectious Pancreatic Necrosis Virus (IPNV), Infectious Salmon Anemia Virus (ISAV), Fish Myocarditis Virus (PMCV), Salmon Alpha Virus (SAV), and Perulan's New Paramotes (Denoted by AGD in Chinese), Candidatus Branchiomonas cysticola (CBc), Microspore Desmozoon lepeophtherii (Des), salmon rickettsiae (P. salmonis, represented by SRS in the table), and salmon kidney (R. salmoninarum, BKD in the table. The results show that each primer pair and probe combination are specific.

Table 27 Specific test results of each primer pair and probe combination

3. Real-time PCR (qPCR)

Use diluted pSGPV plasmids (10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ copy numbers) on a real-time PCR instrument (for example, but not limited to ABI StepOnePlus ^TM ; Applied BioSystem, Life Technologies, California, USA) real-time PCR analysis. Contain a fluorescent molecule 6-carboxyfluorescein (FAM) with 2μl pSGPV plasmid, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.01-2μM probe (5 'and 3' ends of each probe sequence respectively) ) And a fluorescent inhibitor black hole fluorescent inhibitor 1 (BHQ1)), a commercial RT-PCR kit with a total volume of 20 μl (eg, but not limited to, OneStep PrimeScript ^™ RT-PCR Kit; Takara Bio Inc., Japan) Real-time PCR analysis. The real-time PCR program is 42 ° C for 5 minutes, 94 ° C for 10 seconds, and 40 cycles of 94 ° C for 10 seconds and 60 ° C for 30 minutes. Record the results of the fluorescence measurement in the 60 ° C step and calculate. Standard curve for real-time PCR analysis of serially diluted (10-fold) pSGPV plasmids. The results show that at least 10 ² copy number pSGPV plasmids can be detected, and the R ² value of the standard curve of each primer pair and probe combination is greater than 0.99, indicating that the primer pairs and probes of the present invention can be used for Real-time PCR and produce credible results.

The results of the above examples show that the primer pair and the probe of the present invention can be used in the cPCR amplification reaction to detect the presence of salmon parrot virus (SGPV), and have high sensitivity and specificity.

Example 10 Detection of the pathogen P. salmonis

Salmonid rickettsia (P.salmonis) is the pathogen that causes Salmonid Rickettsial Septicaemia (SRS). The 16S ribosomal ribonucleic acid (rRNA) gene (GenBank accession No. AY498634) fragment of P. salmonis is inserted into a selection vector, which can be, but is not limited to, pUC57, pGEM-T to obtain the pSRS plasmid.

1. Traditional polymerase chain reaction

50 l PCR mixture was subjected to a conventional PCR used contained: pSRS ¹⁰⁶ plasmid copy number, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.2μM dNTP and 1.25U Taq DNA polymerase. Amplify the reaction in a thermal cycler (eg, but not limited to PC818, Astec Co. Ltd., Japan), and include an initial cycle of denaturation at 94 ° C for 3 minutes, and 35 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 30 seconds. The amplified product was then analyzed with a 15% polyacrylamide gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) and visualized by ethidium bromide staining.

The results of conventional PCR are shown in Table 28. Each primer pair amplified the correct size fragment of each target sequence, while no target sequence was amplified in the negative control group (results not shown). The results indicate that each primer pair can be used in a traditional PCR amplification reaction to detect the presence of P. salmonis.

Table 28 Results of traditional PCR performed by combining each primer pair and probe

正向引物Forward primer	反向引物Reverse primer	合成片段大小(bp)Synthetic fragment size (bp)
SRS-F1(SEQ ID NO:111)SRS-F1 (SEQ ID NO: 111)	SRS-R1(SEQ ID NO:112)SRS-R1 (SEQ ID NO: 112)	151151
SRS-F2(SEQ ID NO:114)SRS-F2 (SEQ ID NO: 114)	SRS-R2(SEQ ID NO:115)SRS-R2 (SEQ ID NO: 115)	8080
SRS-F3(SEQ ID NO:116)SRS-F3 (SEQ ID NO: 116)	SRS-R3(SEQ ID NO:117)SRS-R3 (SEQ ID NO: 117)	5454
SRS-F4(SEQ ID NO:119)SRS-F4 (SEQ ID NO: 119)	SRS-R4(SEQ ID NO:120)SRS-R4 (SEQ ID NO: 120)	5656
SRS-F5(SEQ ID NO:122)SRS-F5 (SEQ ID NO: 122)	SRS-R5(SEQ ID NO:123)SRS-R5 (SEQ ID NO: 123)	5858

2. Thermal convection polymerase chain reaction (cPCR)

The 50 μl PCR mix contains 5 μl of the RNA of the 16S rRNA gene of P. salmonis (10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ copy number / μl) or the pSRS plasmid (respectively) 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ copies), 0.01-2 μM forward primer, 0.01-2 μM reverse primer, 0.01-2 μM probe (5 ′ end and 3 ′ of each probe sequence A fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescent inhibitory molecule black hole fluorescent inhibitor 1 (BHQ1)), 0.2 μM dNTP, 1X cPCR buffer, and 1-5U Taq DNA polymerase, 1-5U Reverse transcriptase. The PCR mixture is added to a reaction test tube and placed in a thermal convection polymerase chain reaction (cPCR) instrument for a specified period of time (about 30 to 45 minutes). The cPCR instrument was used to detect FAM fluorescence in each sample. The above cPCR analysis test was repeated 8 times (n = 8) to evaluate the sensitivity of each estimated primer to the probe.

The results of the sensitivity test are shown in Table 29. Each primer pair and probe combination can 100% correctly detect 16S rRNA containing 10 ² copies / μl of salmonella rickettsiae (P. salmonii) in the sample. The sensitivity of the gene RNA or 10 ² copy number pSRS plasmids can reach 10 ² copy number / μl and 10 ² copy number.

Table 29 Sensitivity test results of each primer pair and probe combination (n = 8)

In addition, different fish pathogens plasmid (the copy number of ¹⁰⁶ / μl) as a template cPCR analysis of each specific primer pair and probe combination. The cPCR method is as described above. The results of the specificity test are shown in Table 30, and the combination of each primer pair and probe can correctly detect the samples containing salmonella rickettsiae (P. salmonis, represented by SRS in the table) Less than contains fish respiratory and enteroviruses (PRV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia virus (ISAV), fish myocarditis virus (PMCV), salmonella virus (SAV), Peruransin N. perurans (denoted AGD in the table), Candidatus Branchiomonas cysticola (CBc), Microspore Desmozoon lepeophtherii (Des), salmon parrot virus (SGPV), and salmon kidney bacteria (R. salmoninarum, in BKD in the table. The results show that each primer pair and probe combination are specific.

Table 30 Specific test results of each primer pair and probe combination

3. Real-time PCR (qPCR)

Use diluted pSRS plasmids (10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ copy numbers) on a real-time PCR instrument (for example, but not limited to ABI StepOnePlus ^TM ; Applied BioSystem, Life Technologies, California, USA) real-time PCR analysis. Contain a fluorescent molecule 6-carboxyfluorescein (FAM) with 2μl pSRS plasmid, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.01-2μM probe (5 'end and 3' end of each probe sequence respectively) ) And a fluorescent inhibitor Black Hole Fluorescence Inhibitor 1 (BHQ1)), a commercial RT-PCR kit with a total volume of 20 μl (for example, but not limited to, OneStep PrimeScript ^™ RT-PCR Kit; Takara Bio Inc., Japan) Real-time PCR analysis. The real-time PCR program is 42 ° C for 5 minutes, 94 ° C for 10 seconds, and 40 cycles of 94 ° C for 10 seconds and 60 ° C for 30 minutes. Record the results of the fluorescence measurement in the 60 ° C step and calculate. Standard curve for real-time PCR analysis of serially diluted (10-fold) pSRS plasmids. The results show that, at least ¹⁰² pSRS plasmid copy number can be detected, and each primer pair ² values to a standard curve of probe combinations are greater than 0.99 R, represents a primer with a probe of the present invention may be used Real-time PCR and produce credible results.

The results of the above examples show that the primer pair and the probe of the present invention can be used in the cPCR amplification reaction to detect the presence of P. salmonis, and have high sensitivity and specificity.

Example 11 Detection of the pathogen salmonella (R. salmoninarum)

Salmon kidney (R. salmoninarum) is a pathogen that causes bacterial kidney disease (Bacterial kidney disease, BKD) in fish. The 57KD extracellular protein precursor (ECP) gene (GenBank accession No. Z12174) fragment of R. salmoninarum was inserted into a selection vector, which may be, but not limited to, pUC57, pGEM -T to obtain the pBKD plasmid.

1. Traditional polymerase chain reaction

50 l PCR mixture was subjected to a conventional PCR used contained: pBKD ¹⁰⁶ plasmid copy number, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.2μM dNTP and 1.25U Taq DNA polymerase. Amplify the reaction in a thermal cycler (eg, but not limited to PC818, Astec Co. Ltd., Japan), and include an initial cycle of denaturation at 94 ° C for 3 minutes, and 35 cycles of 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 30 seconds. The amplified product was then analyzed with a 15% polyacrylamide gel in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) and visualized by ethidium bromide staining.

The results of traditional PCR are shown in Table 31. Each primer pair amplified the correct size fragment of each target sequence, while no target sequence was amplified in the negative control group (results not shown). The results indicate that each primer pair can be used in a traditional PCR amplification reaction to detect the presence of R. salmoninarum.

Table 31 Results of traditional PCR performed by combining each primer pair and probe

正向引物Forward primer	反向引物Reverse primer	合成片段大小(bp)Synthetic fragment size (bp)
BKD-F1(SEQ ID NO:125)BKD-F1 (SEQ ID NO: 125)	BKD-R1(SEQ ID NO:128)BKD-R1 (SEQ ID NO: 128)	7272
BKD-F2(SEQ ID NO:126)BKD-F2 (SEQ ID NO: 126)	BKD-R1(SEQ ID NO:128)BKD-R1 (SEQ ID NO: 128)	8585
BKD-F3(SEQ ID NO:127)BKD-F3 (SEQ ID NO: 127)	BKD-R1(SEQ ID NO:128)BKD-R1 (SEQ ID NO: 128)	7070
BKD-OF1(SEQ ID NO:130)BKD-OF1 (SEQ ID NO: 130)	BKD-OR1(SEQ ID NO:131)BKD-OR1 (SEQ ID NO: 131)	6565
BKD-OF2(SEQ ID NO:133)BKD-OF2 (SEQ ID NO: 133)	BKD-OR2(SEQ ID NO:134)BKD-OR2 (SEQ ID NO: 134)	125125
BKD-OF3(SEQ ID NO:136)BKD-OF3 (SEQ ID NO: 136)	BKD-OR3(SEQ ID NO:137)BKD-OR3 (SEQ ID NO: 137)	133133

2. Thermal convection polymerase chain reaction (cPCR)

50 μl of the PCR mix contains 5 μl of the RNA of the 57KD extracellular protein precursor (ECP) gene of R. salmoninarum (10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ copies / μl) or pBKD plasmids (respectively 10 ^2, 10 ^3, 10 ^4, 10 ^5, 10 ⁶ copy number), 0.01-2 m forward primer, reverse primer 0.01-2 m, 0.01-2 m probes (probe sequences each 5 The 'end and 3' ends are joined with a fluorescent molecule 6-carboxyfluorescein (FAM) and a fluorescent inhibitory molecule black hole fluorescent inhibitor 1 (BHQ1)), 0.2μM dNTP, 1X cPCR buffer, and 1-5U Taq DNA polymerization Enzyme, 1-5U reverse transcriptase. The PCR mixture is added to a reaction tube and placed in a thermal convection polymerase chain reaction (cPCR) instrument for a specified period of time (approximately 30-45 minutes). The cPCR instrument was used to detect FAM fluorescence in each sample. The above cPCR analysis test was repeated 8 times (n = 8) to evaluate the sensitivity of each estimated primer to the probe.

The results of the sensitivity test are shown in Table 32. The combination of each primer pair and probe can 100% correctly detect the 57KD extracellular protein containing 10 ² copies / μl of R. salmoninarum in the sample. The RNA of the precursor (ECP) gene or the 10 ² copy number pBKD plasmid has a sensitivity of 10 ² copy number / μl and 10 ² copy number.

Table 32 Sensitivity test results of each primer pair and probe combination (n = 8)

In addition, different fish pathogens plasmid (the copy number of ¹⁰⁶ / μl) as a template cPCR analysis of each specific primer pair and probe combination. The cPCR method is as described above. The results of the specificity test are shown in Table 33, and the combination of each primer pair and probe can correctly detect the sample containing salmonella kidney bacteria (R. salmoninarum, represented by BKD in the table), but cannot be detected Contains Fish Respiratory and Enteroviruses (PRV), Infectious Pancreatic Necrosis Virus (IPNV), Infectious Salmon Anemia Virus (ISAV), Fish Myocarditis Virus (PMCV), Salmon A Virus (SAV), and Perulan New Paramorphic Insects (N. perurans, indicated by AGD in the table), Candidatus Branchiomonas cysticola (CBc), microspore insect Desmozoon lepeophtherii (Des), salmon parrot virus (SGPV), and salmon rickettsia (P. salmonis, in Samples in the table are indicated by SRS). The results show that each primer pair and probe combination are specific.

Table 33 Specific test results of each primer pair and probe combination

3. Real-time PCR (qPCR)

Use the diluted pBKD plasmid (10 ¹ , 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ copy numbers) on a real-time PCR instrument (for example, but not limited to ABI StepOnePlus ^TM ; Applied BioSystem, Life Technologies, California, USA) real-time PCR analysis. Contain a fluorescent molecule 6-carboxyfluorescein (FAM) with 2μl pBKD plasmid, 0.01-2μM forward primer, 0.01-2μM reverse primer, 0.01-2μM probe (5 'end and 3' end of each probe sequence respectively) ) And a fluorescent inhibitor black hole fluorescent inhibitor 1 (BHQ1)), a commercial RT-PCR kit with a total volume of 20 μl (eg, but not limited to, OneStep PrimeScript ^™ RT-PCR Kit; Takara Bio Inc., Japan) Real-time PCR analysis. The real-time PCR program is 42 ° C for 5 minutes, 94 ° C for 10 seconds, and 40 cycles of 94 ° C for 10 seconds and 60 ° C for 30 minutes. Record the results of the fluorescence measurement in the 60 ° C step and calculate. Standard curve for real-time PCR analysis of serially diluted (10-fold) pBKD plasmids. The results show that at least 10 ² copy number pBKD plasmids can be detected, and the R ² value of the standard curve of each primer pair and probe combination is greater than 0.99, indicating that the primer pairs and probes of the present invention can be used for Real-time PCR and produce credible results.

The results of the above examples show that the primer pair and probe of the present invention can be used in the cPCR amplification reaction to detect the presence of R. salmoninarum, and have high sensitivity and specificity.

The above detailed description is a specific description of a feasible embodiment of the present invention, but this embodiment is not intended to limit the patent scope of the present invention, and any equivalent implementation or change without departing from the technical spirit of the present invention should include In the patent scope of this case.

Claims

An oligonucleotide pair for detecting fish pathogens includes a first primer and a second primer. The pathogen is selected from the group consisting of: fish respiratory tract and enterovirus (Piscinereovirus, PRV) Infectious pancreatic necrosis virus (IPNV), Infectious salmon anemia virus (ISAV), Piscine myocarditis virus (PMCV), Salmonid alphavirus (SAV) , Neoparamoeba perurans, Candidatus Branchiomonas cysticola (CBc), Microspore Desmozoon lepeophtherii (Des), Salmon Gill Poxvirus (SGPV), Piscirickettsia algae , And salmon kidney bacteria (Renibacterium salmoninarum);

When the pathogen is fish respiratory tract and enterovirus (PRV), the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 1 and SEQ ID NO: 2 , SEQ ID NO: 4 and SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 10, and SEQ ID NO: 11 and SEQ ID NO: 12;

When the pathogen is infectious pancreatic necrosis virus (IPNV), the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 14 and SEQ ID NO: 15 , SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 21, and SEQ ID NO: 23 and SEQ ID NO: 24;

Where the pathogen is an infectious salmon anemia virus (ISAV), the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 26 and SEQ ID NO: 27, SEQ ID NO: 29 and SEQ ID NO: 30, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 36, and SEQ ID NO: 37 and SEQ ID NO: 38;

When the pathogen is fish myocarditis virus (PMCV), the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 39 and SEQ ID NO: 40, SEQ ID NO: 42 and SEQ ID: NO: 43, SEQ ID: NO: 45 and SEQ ID: NO: 46, SEQ ID: NO: 48 and SEQ ID: NO: 49, SEQ ID: NO: 51 and SEQ ID: NO: 49, and SEQ ID: NO : 52 and SEQ ID NO: 49;

When the pathogen is Salmon Alpha Virus (SAV), the sequence combination of the first primer and the second primer is selected from the group consisting of SEQ ID NO: 53 and SEQ ID NO: 54, SEQ ID NO: 56 and SEQ ID NO: 57, SEQ ID NO: 59 and SEQ ID NO: 60, SEQ ID NO: 62 and SEQ ID NO: 63, and SEQ ID NO: 65 and SEQ ID NO: 66;

When the pathogen is N. perurans, the sequence combination of the first primer and the second primer is selected from the group consisting of SEQ ID NO: 68 and SEQ ID NO: 69, SEQ ID NO: 71 and SEQ ID NO: 72, SEQ ID NO: 74 and SEQ ID NO: 75, and SEQ ID NO: 77 and SEQ ID NO: 78;

When the pathogen is Ca.B. cysticola, the sequence combination of the first primer and the second primer is selected from the group consisting of SEQ ID NO: 80 and SEQ ID NO: 81, and SEQ ID NO: 83 and SEQ ID NO: 84;

When the pathogen is D. lepeophtherii, the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 86 and SEQ ID NO: 87, SEQ ID NO: 89 and SEQ ID NO: 90, SEQ ID NO: 92 and SEQ ID NO: 93, SEQ ID NO: 95 and SEQ ID NO: 97, and SEQ ID NO: 96 and SEQ ID NO: 97;

When the pathogen is salmon parrot virus (SGPV), the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 99 and SEQ ID NO: 100, SEQ ID NO: 102 and SEQ ID NO: 103, SEQ ID NO: 105 and SEQ ID NO: 106, and SEQ ID NO: 108 and SEQ ID NO: 109;

When the pathogen is P. salmonis, the sequence combination of the first primer and the second primer is selected from the group consisting of SEQ ID NO: 111 and SEQ ID NO: 113, SEQ ID NO: 114 and SEQ ID NO: 115, SEQ ID NO: 116 and SEQ ID NO: 117, SEQ ID NO: 119 and SEQ ID NO: 120, and SEQ ID NO: 122 and SEQ ID NO: 123 ;as well as

When the pathogen is R. salmoninarum, the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 126 and SEQ ID NO: 128, SEQ ID NO: 127 and SEQ ID NO: 128, SEQ ID NO: 130 and SEQ ID NO: 131, SEQ ID NO: 133 and SEQ ID NO: 134, and SEQ ID NO: 136 and SEQ ID NO: 137.
The oligonucleotide pair according to claim 1, further comprising an oligonucleotide probe having an oligonucleotide probe sequence, an appended to the oligonucleotide A fluorescent molecule at a first position on the probe, and a fluorescent inhibitor molecule at a second position attached to the oligonucleotide probe.
The oligonucleotide pair of claim 2, wherein the pathogen is fish respiratory tract and enterovirus (PRV), and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 1 and SEQ ID NO: 2, the oligonucleotide probe sequence is a sequence between the pathogen fish respiratory tract and enterovirus ( (PRV) 13 to 25 base pair oligonucleotides between 71 to 95 nucleotides of the L1 segment gene;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 4 and SEQ ID NO: 2, the oligonucleotide probe sequence is a sequence between the pathogen fish respiratory tract and enterovirus ( (PRV) 13 to 35 base pair oligonucleotides between the 52nd to 95th nucleotides of the L1 segment gene;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 4 and SEQ ID NO: 5, the oligonucleotide probe sequence is a sequence between the pathogen fish respiratory tract and enterovirus ( (PRV) 13 to 35 base pair oligonucleotides between the 52nd to 100th nucleotides of the L1 segment gene;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 6 and SEQ ID NO: 7, the oligonucleotide probe sequence is a sequence between the pathogen fish respiratory tract and enterovirus ( (PRV) 13 to 35 base pair oligonucleotides between 343 and 385 nucleotides of the Sigma 3 protein gene;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 9 and SEQ ID NO: 10, the oligonucleotide probe sequence is a sequence between the pathogen fish respiratory tract and enterovirus ( PRV) 13 to 35 base pair oligonucleotides from 350 to 380 nucleotides of the Sigma 3 protein gene; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 11 and SEQ ID NO: 12, the oligonucleotide probe sequence is a sequence between the pathogen fish respiratory tract and enterovirus ( (PRV) 13 to 35 base pair oligonucleotides between the 280th to 315th nucleotides of the L1 segment gene.
The oligonucleotide pair of claim 2, wherein the pathogen is fish respiratory tract and enterovirus (PRV), and

The sequence combination of the first primer and the second primer is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 2, and SEQ ID When NO: 4 and SEQ ID NO: 5, the oligonucleotide probe sequence is shown as SEQ ID NO: 3;

When the sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 6 and SEQ ID NO: 7, and SEQ ID NO: 9 and SEQ ID NO: 10, The oligonucleotide probe sequence is shown in SEQ ID NO: 8; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 11 and SEQ ID NO: 12, the oligonucleotide probe sequence is shown in SEQ ID NO: 13.
The oligonucleotide pair of claim 2, wherein the pathogen is infectious pancreatic necrosis virus (IPNV), and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 14 and SEQ ID NO: 15, the oligonucleotide probe sequence is an infectious pancreatic necrosis virus between the pathogen ( (IPNV) 13 to 35 base pair oligonucleotides between 2010 to 2057 nucleotides of VP5 and the polyprotein gene;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 17 and SEQ ID NO: 18, the oligonucleotide probe sequence is an infectious pancreatic necrosis virus infectious with the pathogen ( IPNV) oligonucleotides of 13 to 35 base pairs between the 2345th to 2408th nucleotides of the VP5 of the polyprotein gene;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 20 and SEQ ID NO: 21, the oligonucleotide probe sequence is an infectious pancreatic necrosis virus between the pathogen ( IPNV) oligonucleotides of 13 to 35 base pairs between the 2533th to 2580th nucleotides of the VP5 of the polyprotein gene; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 23 and SEQ ID NO: 24, the oligonucleotide probe sequence is an infectious pancreatic necrosis virus infectious with the pathogen ( (IPNV) oligos of 13 to 35 base pairs between the VP5 of the polyprotein gene and the 2775th to 2818th nucleotides of the polyprotein gene.
The oligonucleotide pair of claim 2, wherein the pathogen is infectious pancreatic necrosis virus (IPNV), and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 14 and SEQ ID NO: 15, the oligonucleotide probe sequence is shown in SEQ ID NO: 16;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 17 and SEQ ID NO: 18, the oligonucleotide probe sequence is shown in SEQ ID NO: 19;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 20 and SEQ ID NO: 21, the oligonucleotide probe sequence is shown in SEQ ID NO: 22; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 23 and SEQ ID NO: 24, the oligonucleotide probe sequence is shown in SEQ ID NO: 25.
The oligonucleotide pair of claim 2, wherein the pathogen is an infectious salmon anemia virus (ISAV), and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 26 and SEQ ID NO: 27, the oligonucleotide probe sequence is an infectious salmon anemia virus (ISAV) between the pathogen ) Open reading frame (Open reading frame, ORF) 1 and ORF2 gene between 282 to 328 nucleotides 13 to 35 base pairs of oligonucleotides;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 29 and SEQ ID NO: 30, the oligonucleotide probe sequence is an infectious salmon anemia virus (ISAV) between the pathogen ) 13 to 35 base pair oligonucleotides between the 79th to 121st nucleotides of the ORF1 and ORF2 genes;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 32 and SEQ ID NO: 33, the oligonucleotide probe sequence is an infectious salmon anemia virus (ISAV) between the pathogen ) 13 to 30 base pair oligonucleotides between the 244th to 273rd nucleotides of the ORF1 and ORF2 genes;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 35 and SEQ ID NO: 36, the oligonucleotide probe sequence is an intervening pathogenic infectious salmon anemia virus (ISAV) ) 13 to 35 base pair oligonucleotides between the 237th to 272nd nucleotides of the ORF1 and ORF2 genes; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 37 and SEQ ID NO: 38, the oligonucleotide probe sequence is an infectious salmon anemia virus (ISAV) between the pathogen ) 13 to 35 base pair oligonucleotides between the 235th to 286th nucleotides of the ORF1 and ORF2 genes.
The oligonucleotide pair of claim 2, wherein the pathogen is an infectious salmon anemia virus (ISAV), and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 26 and SEQ ID NO: 27, the oligonucleotide probe sequence is shown in SEQ ID NO: 28;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 29 and SEQ ID NO: 30, the oligonucleotide probe sequence is shown in SEQ ID NO: 31; and

The sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 35 and SEQ ID NO: 36, and SEQ ID When NO: 37 and SEQ ID NO: 38, the oligonucleotide probe sequence is shown as SEQ ID NO: 34.
The oligonucleotide pair of claim 2, wherein the pathogen is fish myocarditis virus (PMCV), and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 39 and SEQ ID NO: 40, the oligonucleotide probe sequence is a sequence between the pathogen fish myocarditis virus (PMCV) 13 to 35 base pair oligonucleotides between the 6266th to 6306th nucleotides of the genome;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 42 and SEQ ID NO: 43, the oligonucleotide probe sequence is a sequence between the pathogen fish myocarditis virus (PMCV) 13 to 35 base pair oligonucleotides between 5632 to 5672 nucleotides in the genome;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 45 and SEQ ID NO: 46, the oligonucleotide probe sequence is a sequence between the pathogen fish myocarditis virus (PMCV) 13 to 35 base pair oligonucleotides between 5693 to 5747 nucleotides in the genome;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 48 and SEQ ID NO: 49, the oligonucleotide probe sequence is a sequence between the pathogen fish myocarditis virus (PMCV) 13 to 35 base pair oligonucleotides between 6294 to 6331 nucleotides in the genome;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 51 and SEQ ID NO: 49, the oligonucleotide probe sequence is a sequence between the pathogen fish myocarditis virus (PMCV) 13 to 35 base pair oligonucleotides between 6271 to 6331 nucleotides in the genome; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 52 and SEQ ID NO: 49, the oligonucleotide probe sequence is a sequence between the pathogen fish myocarditis virus (PMCV) 13 to 35 base pair oligonucleotides between the 6290th and 6331st nucleotides of the genome.
The oligonucleotide pair of claim 2, wherein the pathogen is fish myocarditis virus (PMCV), and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 39 and SEQ ID NO: 40, the oligonucleotide probe sequence is shown in SEQ ID NO: 41;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 42 and SEQ ID NO: 43, the oligonucleotide probe sequence is shown in SEQ ID NO: 44;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 45 and SEQ ID NO: 46, the oligonucleotide probe sequence is shown in SEQ ID NO: 47; and

The sequence combination of the first primer and the second primer is selected from the group consisting of SEQ ID NO: 48 and SEQ ID NO: 49, SEQ ID NO: 51 and SEQ ID NO: 49, and SEQ ID When NO: 52 and SEQ ID NO: 49, the oligonucleotide probe sequence is shown as SEQ ID NO: 50.
The oligonucleotide pair of claim 2, wherein the pathogen is salmonella virus (SAV), and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 53 and SEQ ID NO: 54, the oligonucleotide probe sequence is a sequence between the pathogen Salmon Alpha Virus (SAV) 13 to 35 base pair oligonucleotides between 611 to 653 nucleotides of the genome;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 56 and SEQ ID NO: 57, the oligonucleotide probe sequence is a sequence between the pathogen Salmon Alpha Virus (SAV) 13 to 35 base pair oligonucleotides between 145 to 181 nucleotides of the genome;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 59 and SEQ ID NO: 60, the oligonucleotide probe sequence is a sequence between the pathogen Salmon Alpha Virus (SAV) 13 to 35 base pair oligonucleotides between 2691 to 2761 nucleotides in the genome;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 62 and SEQ ID NO: 63, the oligonucleotide probe sequence is a sequence between the pathogen Salmon Alpha Virus (SAV) 13 to 35 base pair oligonucleotides between 1032 to 1075 nucleotides of the genome; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 65 and SEQ ID NO: 66, the oligonucleotide probe sequence is a sequence between the pathogen Salmon Alpha Virus (SAV) 13 to 35 base pair oligonucleotides between 969 to 1011 nucleotides of the genome.
The oligonucleotide pair of claim 2, wherein the pathogen is salmonella virus (SAV), and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 53 and SEQ ID NO: 54, the sequence of the oligonucleotide probe is shown in SEQ ID NO: 55;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 56 and SEQ ID NO: 57, the oligonucleotide probe sequence is shown in SEQ ID NO: 58;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 59 and SEQ ID NO: 60, the oligonucleotide probe sequence is shown in SEQ ID NO: 61;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 62 and SEQ ID NO: 63, the oligonucleotide probe sequence is shown in SEQ ID NO: 64; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 65 and SEQ ID NO: 66, the oligonucleotide probe sequence is shown in SEQ ID NO: 67.
The oligonucleotide pair according to claim 2, wherein the pathogen is N. perurans, and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 68 and SEQ ID NO: 69, the sequence of the oligonucleotide probe is an intercalary amoeba between the pathogen Perulan (N. perurans) 18S ribosomal ribonucleic acid (ribosomal RNA, rRNA) gene between 462 to 520th nucleotides 13 to 35 base pairs of oligonucleotides;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 71 and SEQ ID NO: 72, the oligonucleotide probe sequence is a new paramyxoplasma that is between the pathogen Perulan (N. perurans) oligonucleotides of 13 to 35 base pairs between the 355th to 405th nucleotides of the 18S rRNA gene;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 74 and SEQ ID NO: 75, the oligonucleotide probe sequence is a new paramyxoplasma intervening with the pathogen Perulan (N. perurans) oligonucleotides of 13 to 35 base pairs between the 234th to 281st nucleotides of the 18S rRNA gene; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 77 and SEQ ID NO: 78, the oligonucleotide probe sequence is a new paraamoeba between the pathogen Perulan (N. perurans) an oligonucleotide of 13 to 24 base pairs between the 357th to 380th nucleotides of the 18S rRNA gene.
The oligonucleotide pair according to claim 2, wherein the pathogen is N. perurans, and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 68 and SEQ ID NO: 69, the oligonucleotide probe sequence is shown in SEQ ID NO: 70;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 71 and SEQ ID NO: 72, the oligonucleotide probe sequence is shown in SEQ ID NO: 73;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 74 and SEQ ID NO: 75, the oligonucleotide probe sequence is shown in SEQ ID NO: 76; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 77 and SEQ ID NO: 78, the oligonucleotide probe sequence is shown in SEQ ID NO: 79.
The oligonucleotide pair of claim 2, wherein the pathogen is Ca.B. cysticola, and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 80 and SEQ ID NO: 81, the oligonucleotide probe sequence is a 16S between the pathogen Ca.B.cysticola Ribosomal ribonucleic acid (ribosomal RNA, rRNA) gene 13 to 35 base pair oligonucleotides between 1068 to 1125 nucleotides; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 83 and SEQ ID NO: 84, the oligonucleotide probe sequence is a 16S between the pathogen Ca.B.cysticola An oligonucleotide of 13 to 30 base pairs between the 1212th to 1241st nucleotides of the rRNA gene.
The oligonucleotide pair of claim 2, wherein the pathogen is Ca.B. cysticola, and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 80 and SEQ ID NO: 81, the oligonucleotide probe sequence is shown in SEQ ID NO: 82; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 83 and SEQ ID NO: 84, the oligonucleotide probe sequence is shown in SEQ ID NO: 85.
The oligonucleotide pair of claim 2, wherein the pathogen is D. lepeophtherii, and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 86 and SEQ ID NO: 87, the oligonucleotide probe sequence is a sequence between the pathogen Microsporidium D. lepeophtherii Oligonucleotides of 13 to 35 base pairs between the 967th to 1010th nucleotides of the 16S ribosomal ribonucleic acid (ribosomal RNA, rRNA) gene;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 89 and SEQ ID NO: 90, the oligonucleotide probe sequence is a sequence between the pathogen Microsporidium D. lepeophtherii Oligonucleotides of 13 to 35 base pairs between the 1246th to 1285th nucleotides of the 16S rRNA gene;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 92 and SEQ ID NO: 93, the oligonucleotide probe sequence is a sequence between the pathogen Microsporidium D. lepeophtherii Oligonucleotides of 13 to 35 base pairs between the 1366th to 1414th nucleotides of the 16S rRNA gene;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 95 and SEQ ID NO: 97, the oligonucleotide probe sequence is a sequence between the pathogen Microsporidium D. lepeophtherii An oligonucleotide of 13 to 35 base pairs between the 815th to 859th nucleotides of the 16S rRNA gene; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 96 and SEQ ID NO: 97, the oligonucleotide probe sequence is a sequence between the pathogen Microsporidium D. lepeophtherii An oligonucleotide of 13 to 35 base pairs between the 817th to 859th nucleotides of the 16S rRNA gene.
The oligonucleotide pair of claim 2, wherein the pathogen is D. lepeophtherii, and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 86 and SEQ ID NO: 87, the oligonucleotide probe sequence is shown in SEQ ID NO: 88;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 89 and SEQ ID NO: 90, the oligonucleotide probe sequence is shown in SEQ ID NO: 91;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 92 and SEQ ID NO: 93, the oligonucleotide probe sequence is shown in SEQ ID NO: 94; and

The sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 95 and SEQ ID NO: 97, and SEQ ID NO: 96 and SEQ ID NO: 97, The oligonucleotide probe sequence is shown in SEQ ID NO: 98.
The oligonucleotide pair of claim 2, wherein the pathogen is salmon parrot virus (SGPV), and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 99 and SEQ ID NO: 100, the oligonucleotide probe sequence is a sequence between the pathogen salmon parrot virus (SGPV) 13 to 35 base pair oligonucleotides between 99272 to 99318 nucleotides in the genome of

When the sequence combination of the first primer and the second primer is SEQ ID NO: 102 and SEQ ID NO: 103, the oligonucleotide probe sequence is a sequence between the pathogen salmon parrot virus (SGPV) 13 to 27 base pair oligonucleotides between 123213 to 123239 nucleotides of the genome of

When the sequence combination of the first primer and the second primer is SEQ ID NO: 105 and SEQ ID NO: 106, the oligonucleotide probe sequence is a sequence between the pathogen salmon parrot virus (SGPV) 13 to 30 base pair oligonucleotides between 123202 to 123232 nucleotides in the genome of the genome; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 108 and SEQ ID NO: 109, the oligonucleotide probe sequence is a sequence between the pathogen salmon parrot virus (SGPV) 13 to 35 base pair oligonucleotides between the 123556th to 123610th nucleotides of the genome.
The oligonucleotide pair of claim 2, wherein the pathogen is salmon parrot virus (SGPV), and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 99 and SEQ ID NO: 100, the oligonucleotide probe sequence is shown in SEQ ID NO: 101;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 102 and SEQ ID NO: 103, the oligonucleotide probe sequence is shown in SEQ ID NO: 104;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 105 and SEQ ID NO: 106, the oligonucleotide probe sequence is shown in SEQ ID NO: 107; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 108 and SEQ ID NO: 109, the oligonucleotide probe sequence is shown in SEQ ID NO: 110.
The oligonucleotide pair of claim 2, wherein the pathogen is P. salmonis, and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 111 and SEQ ID NO: 112, the oligonucleotide probe sequence is an intermediate between the pathogen salmon rickettsiae (P .salmonis) 16S ribosomal ribonucleic acid (ribosomal RNA, rRNA) gene 13 to 35 base pair oligonucleotides between 952 to 1065 nucleotides;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 114 and SEQ ID NO: 115, the oligonucleotide probe sequence is an intermediate between the pathogen salmon rickettsiae (P .salmonis) oligonucleotides of 13 to 35 base pairs between the 1014th to 1050th nucleotides of the 16S rRNA gene;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 116 and SEQ ID NO: 117, the oligonucleotide probe sequence is an intermediate between the pathogen salmon rickettsiae (P .salmonis) 13S-17 base pair oligonucleotides between the 315th to 331rd nucleotides of the 16S rRNA gene;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 119 and SEQ ID NO: 120, the oligonucleotide probe sequence is an intermediate between the pathogen salmon rickettsiae (P .salmonis) oligonucleotides of 13 to 17 base pairs between the 529th to 545th nucleotides of the 16S rRNA gene; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 122 and SEQ ID NO: 123, the oligonucleotide probe sequence is an intermediate between the pathogen salmon rickettsiae (P .salmonis) 13S to 20 base pair oligonucleotides between 1181 to 1200 nucleotides of the 16S rRNA gene.
The oligonucleotide pair of claim 2, wherein the pathogen is P. salmonis, and

The sequence combination of the first primer and the second primer is selected from the group consisting of: SEQ ID NO: 111 and SEQ ID NO: 112, and SEQ ID NO: 114 and SEQ ID NO: 115. The oligonucleotide probe sequence is shown in SEQ ID NO: 113;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 116 and SEQ ID NO: 117, the oligonucleotide probe sequence is shown in SEQ ID NO: 118;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 119 and SEQ ID NO: 120, the oligonucleotide probe sequence is as shown in SEQ ID NO: 121; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 122 and SEQ ID NO: 123, the oligonucleotide probe sequence is shown in SEQ ID NO: 124.
The oligonucleotide pair according to claim 2, wherein the pathogen is R. salmoninarum, and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 125 and SEQ ID NO: 128, the oligonucleotide probe sequence is an intervening pathogen R. salmoninarum ) Of 13 to 31 base pair oligonucleotides between the 1035th to the 1065th nucleotides of the 57KD extracellular protein precursor (ECP) gene;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 126 and SEQ ID NO: 128, the oligonucleotide probe sequence is an intervening pathogen R. salmoninarum ) An oligonucleotide of 13 to 35 base pairs between 1024 to 1065 nucleotides of the 57KD extracellular protein precursor (ECP) gene;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 127 and SEQ ID NO: 128, the oligonucleotide probe sequence is an intervening pathogen R. salmoninarum ) An oligonucleotide of 13 to 29 base pairs between the 1037th to 1065th nucleotides of the 57KD extracellular protein precursor (ECP) gene;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 130 and SEQ ID NO: 131, the oligonucleotide probe sequence is an intervening pathogen R. salmoninarum ) An oligonucleotide of 13 to 25 base pairs between the 782th to 806th nucleotides of the 57KD extracellular protein precursor (ECP) gene;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 133 and SEQ ID NO: 134, the oligonucleotide probe sequence is an intervening pathogen R. salmoninarum ) An oligonucleotide of 13 to 35 base pairs between the 880th to 964th nucleotides of the 57KD extracellular protein precursor (ECP) gene; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 136 and SEQ ID NO: 137, the oligonucleotide probe sequence is an intermediate of the pathogen salmonella ) 13 to 35 base pair oligonucleotides between the 646th to 738th nucleotides of the 57KD extracellular protein precursor (ECP) gene.
The oligonucleotide pair according to claim 2, wherein the pathogen is R. salmoninarum, and

The sequence combination of the first primer and the second primer is selected from the group consisting of SEQ ID NO: 125 and SEQ ID NO: 128, SEQ ID NO: 126 and SEQ ID NO: 128, and SEQ ID When NO: 127 and SEQ ID NO: 128, the oligonucleotide probe sequence is shown as SEQ ID NO: 129;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 130 and SEQ ID NO: 131, the oligonucleotide probe sequence is shown in SEQ ID NO: 132;

When the sequence combination of the first primer and the second primer is SEQ ID NO: 133 and SEQ ID NO: 134, the oligonucleotide probe sequence is shown in SEQ ID NO: 135; and

When the sequence combination of the first primer and the second primer is SEQ ID NO: 136 and SEQ ID NO: 137, the oligonucleotide probe sequence is shown in SEQ ID NO: 138.